Lymphokine production and purification

Production and purification of lymphokine

Full description

Background of the invention:

This invention relates to the production of a protein that has the ability to stimulate the growth and differentiation of primate hematopoietic cells. It also relates in particular to the protein CSF colony stimulating factor. This invention also works, in one of its forms, to prepare a method for producing a protein by means of DNA processes that artificially enter the cell in order to change the genetic type and climate of the cell, and to coil along the length of the natural DNA. The invention also relates to genetic carriers containing a gene in order to modify that gene for the aforementioned protein. The invention also relates to microorganisms and cell lines transformed transformed with the aforementioned vectors. It also relates to the CSF protein formed.

The different types of cells found in the blood are all derived from bone marrow stem cells that make up blood cells. These bone marrow cells have two functions: (1) - They reconstitute themselves and thus maintain the quality of marrow cells in the body, (2) - They work to prepare progeny cells authorized to differentiate into mature blood cell types. The cells mandated to differentiate into the special pathway that makes up blood cells are called progenitor cells. The progenitor cells of T lymphocytes, B lymphocytes, granulocytes, red blood cells, and platelets have been studied.

And eosinophils, in addition to progenitor cells

Early cells that can independently give rise to many types of mature cells - experimentally, either internally or externally Dexter, TM 1983 J. pathology 141 415-433 (vitro). It has been determined externally that the proliferation and/or diversity of each type of progenitor cell depends on special factors derived from different sources. For example, the last progenitor cells of red blood cells require the factor erythropoietin (a factor that helps form red blood cells). The factors required for surval life, and the proliferation and diversity of myeloid progenitor cells mandated to form mature granulocytes that are neutral to the eosin pigment, granulocytes, monocytes, and macrophages, these factors are called protein colony stimulating factors.

CSFs (colony stimulating factors)-

CSF activity has been studied extensively in mice. It is found in most adult mice. However, formulations containing CSF activity have been obtained from different tissues by different methods that appear to differ in their biochemical properties. Also, the structural relationships between the different factors are still unknown. Moreover, CSF activity appears to be involved in more than one step in the development of granulocytes and macrophages. Again, it is not certain whether a single factor is responsible for all the observed activities or whether a different factor. Works on every step

(947-957 56 Burgess, A. and Metcalf, D. 1980 Blood).

CSF activity in humans has been obtained from placenta,

Some fetal tissues, macrophage cells, and activated T cells. A line of T cells (specific to Mo mice) has been created that induces one or more activities

Strong CSF from a patient with T-cell type 1068-1072 (leukaemic reticuloen dotheliosis) (Golde et al 1978 Blood

The ability of CSF activity to stimulate the production of granulocytes and macrophages indicates that pharmaceutical compositions that have CSF activity are clinically useful when increasing the production of this type of cell (myeloid) is required. In fact, it has been shown that many patients who are severely affected by high levels of granulocytes that circulate normally in the blood have tumors that are very abundant in the CSF. In one case, once the tumor was removed, there was a rapid decrease in the number of granulocytes towards the normal level, which strongly confirms that CSF can be useful; In regulating the numbers of circulating granulocytes in the blood (1983) 600 61 and Golde, D. Blood J., Hocking, W., Goodman) In particular, CSF formulations are clinically useful in treating myelo-suppression resulting from chemotherapeutical or radiation therapy for cancer. In addition, CSF formulations are useful in treating infections because CSF can increase or activate the number of cells and/or monocytes.

There are many different types of known CSF activities including,

CSF granulocytes and macrophages (GM-CSF) and multi-CSF, and the present invention relates to GM-CSF. CSF proteins are known from various animal sources. However, the present invention relates to the first GM-CSF, and more specifically to human GM-CSF and monkey GM-CSF.

ape.

The biological and biochemical identification of formulations with CSF activity and the study of these formulations in the clinical setting has become difficult today due to the scarcity and impurity of human CSF formulations and/or other CSF formulations. It is expected that it will be required to identify the protein or proteins responsible for CSF activity. Furthermore, a source, preferably human, of such CSF that can easily be supplied is required

These proteins are in sufficient quantities and purity for bioassay

Chemicals for use as therapeutic agents.

Recently, there are developed (methods) of molecular cloning that have made it possible to combine an uncleotide nucleotide sequence - which is included in the internal code of a protein - and such a protein is produced in quantity using a carrier system specific to the host Maniatis, T. et.MolecularCloning-A Laboratory Manual (1982 - Cold Spring Harbor, NY) The conjugation methods used today can be regrouped into three well-known separation and purification methods, and the conjugation methods used today can be grouped into three general groups: (1) Methods that depend on knowing the structure of the protein, such as its amino acid sequence, (2) Methods that depend on identifying the protein that has been copied by a gene that is combined using an antibiotic specific to this protein, (3) Methods that depend on identifying the types of RNA that can Transcribed to produce the protein or activity that codes for it - the gene of interest.

It becomes difficult to apply each of these bands when the protein of interest, such as CSF protein, is available in a very small amount. It is also difficult to obtain a sufficient amount of purified protein, and it is difficult to determine the amino acid sequence or even the sequences.

Molecular protein. Likewise, identification of the copied protein with an antibody conjugation is preferable by using a special, individual, high-level, multipolar antiserum. Such an antiserum cannot be obtained without the presence of quantities of pure protein (antigen). A monopolar antibody provides an alternative, but the required antibody can also be difficult to obtain in the absence of the appropriate antigen, and such a monopolar antibody may not react with the protein in the form in which the protein is copied by providing host-specific carrier systems. Genetic and climate change in the cell. Ultimately, transferring RNA species to produce a recognizable protein or activity requires that the RNA in question be present in the RNA source in sufficient abundance to provide a reliable protein or activity signal. In general, the relative tension of RNA is included in the internal code for a particular protein = protein availability, so that a rare protein is often coded by a rare mRNA rarc.

The Mo cell line has been used both as a starting material to purify human CSFs and to identify corresponding messenger RNAs. However, even with this relatively good source of CSF activity it has proven very difficult to isolate sufficient protein for compositional studies.

U.S. Patent No. 4,438,032 discussed partial purification of CSF and practical general notations for genetic and climatic type variation on the cell that allegedly constitutes this. However, it has been stated that the purified material likely contains less than 40% CSF and the process of changing the genetic and climate type on the cell is claimed to be dependent.

contains structural information that did not exist before the present invention. British patent (UK) A2.092159 relates to a process of separating human CSF from hybridoma cells. However, it is not clear to which CSF this request relates. Hybrid cells could not be deposited, and therefore this statement could not be verified.

To overcome the problems associated with combining the nucleotide sequence, which is part of the internal code of a rare protein such as CSF, using the methods described above, a new method was developed. This method only requires that the gene output or activity can be measured accurately. Suitable methods for assessing GM-CSF are described in Example 2 next.

General description of the invention:

In the first aspect of the present invention, it overcomes the problems of the previous avt technology and provides a ready source of a protein that has GM-CSF activity using a synthetic DNA method for the cell in order to change the genetic type and climate of the cell and wrap the length of the natural DNA. According to the present invention, a new combination method is used that only requires testing the activity of GM-CSF. The present invention also provides a cDNA gene carrying the internal code for a protein having GM-CSF activity (i.e., GM-CSF/cDNA). A host-specific microorganism or cell line transformed by a carrier of a genetic and climate-type variation on the cell containing such GM-CSF/cDNA, such as Genetic and climate change carriers on the cell and a method for producing GM-CSF protein by copying the said GM-CSF/cDNA by culturing a microorganism or cell line. Since the GM-CSF protein is formed from a clone pole according to the present invention, we can confirm that it is a protein that has GM-CSF activity. In addition, it includes

The invention is a method for preparing and isolating a transformation carrier containing GM-CSF/cDNA, and the said method includes:

RNA preparation from GM-CSF-forming cell;

An RNA messenger carrier (polyadenylated messenger RNA) is prepared from said RNA;

- Preparation of cDNA from a single strand of the mentioned RNA carrier;

- Conversion of single-stranded cDNA into double-stranded cDNA;

- Inserting double-stranded cDNA into transformation carriers and bacteria are transformed by the aforementioned carrier to form colonies;

- Capturing pools of approximately 200-500 colonies each and isolating an RNA Plasmid from each spot.

- Transfer of plasmid DNA into host cells suitable for CSF protein transcription;

- Culturing the transferred cells and examining the float for GM-CSF activity in making the selection of positive GM-CSF spots and examining the colonies used in making the spot to identify the colony that has CSF activity.

The GM-CSF proteins of this invention are growth and diversity hormones for the cells of the myeloid system. For example, it is prescribed for use clinically to treat myelosuppression, especially (symptomatic) white granulocytopenia resulting from chemotherapy or radiotherapy for cancer.

Brief explanation of the drawings:

Figure 1: Illustrates DNA sequences containing the internal code for the GM-CSF protein according to the present invention. The DNA sequence codes for one type of human GM-CSF called CSF-Thr. there

Another allele contains the code for the identical product except that Thr at position 100 is replaced by (CSF-lle). The changes shown above the human sequence are due to differences in the DNA sequence that contains the code for GM-CSF of the gibbon ape (GM-CSF of the gibbon ape). Reduction of deduced amino acid sequences is also shown.

Figure 2: Schematic showing the preparation of pTPL plasmid from pAdD26SvpA(3) plasmid.

Figure 3: Continuation diagram from Figure 2 showing the preparation of plasmid p91023 from plasmid pTPL.

Figure 4: Continuation diagram of Figure 3 showing plasmid p91023(B).

Figure 6: Schematic representation of pTAF.C-.185R carrier.

Figure 7: Layout representation of a 14-AJ holder.

Detailed description:

The following definitions are provided to facilitate understanding of this case. To the extent that the definitions differ from the meaning that revolves within the technology, the definitions are below for comparison.

Amplification: means the process by which the cells that make up the gene are replicated within the chromosomal DNA.

.

GM-CSF: This is a biological activity known as the tests as described here.

GM-CSF protein: It is a protein from a primary source with activity, GM-CSF

For the purposes of the present invention the term protein

GM-CSF includes a modified GM-CSF protein,

Similar profile of GM-CSF protein GM-CSF protein is preceded by a MET residue.

Down: Downstream means the direction heading towards the end 3 in

Nucleotide sequence.

An enhancer: means a nucleotide sequence that can strengthen gene transfer

Regardless of the position of the helper relative to the gene or the direction of the sequence.

Gene: means deoxyribonucleotide sequence

It includes the code for a specific protein. For purposes here, the code will not include non-transcribed terminal regions such as RNA transfer initiation signals, polyadenylation sites, promoters or enhancers.

Ligation: is the process of forming a phosphodiester bond

phosphldiester between ends 15 and 13 of two strands of DNA.

This can be done by several known enzymatic methods including broad-end ligation by T4 ligase.

Orientation: means the direction of nucleotides in the DNA sequence. direction

An inverted (inverted) DNA sequence is one in which the direction of 15 to 13 in the sequence with respect to another sequence is inverted when compared to the point of return in the DNA from which the sequence was obtained. These reference points can include the direction of transformation of DNA sequences assigned to the DNA source or

Reproductive origin (reproduction) Reproducible carriers containing the sequence.

Transcription: means the synthesis of RNA from a copy of DNA.

Transformation: means changing the genetic type of a cell by capture

Cellular exogenous DNA. The shift can in some cases be determined by a change in climate type

The converted cells are called transformers. Pre-metamorphic cells are referred to as mother cells.

Translation: means the synthesis of a polypeptide from a message carrier RNA. GM-colony-stimulating matrix (GM-CSF) can be derived from a number of cellular sources including a conditioned medium of mononuclear cells found in peripheral blood, lung, placental tissue and bone marrow. Marrow, urine from patients with anemic anemia, serum, normal and neoplastic cells of the T-lymphocyte type and the mononuclear phagocyte lineage. One cell line that is GM-CSF is the Mo cell line precipitated by and available from ATCC under code number CRL8066. The CSF formed by this cell line is, of course, human CSF. One of the sources of GM-CSF in Al-Aboun is a T-cell line called 144-UCD MLA, precipitated B, and available from ATCC under the code number 9370 HB, precipitated on September 29, 1983 AD.

For the purpose of separating GM-CSF Isolate according to the present invention, a new method was used. We only require a method for examining GM-CSF activity. First: it is done

Identify cells that produce GM-CSF activity, such as T-lymphocyte cells (or other sources such as those mentioned above). Then the cell's mRNA is immunoprecipitated, preferably using T-lymph0cyte cells. In this case, the mRNA bound to the cell membrane containing the mRNA for lymphokines is separated from the free mRNA in the cells. It is believed that this separation strengthens the collected mRNA 5-10 times for lymphokine cascades and also reduces the effort required to recognize the required GM-CSF. Polyadenylated RNA carrying the message is then prepared by chromatography over oligo dt cellulose.

cDNA is prepared from mRNA using a suitable carrier for transfer to the host to modify the desired protein that has GM-CSF activity. First, the cDNA strand is prepared using typical methods using the prepared mRNA. The RNA/cDNA hybrid is then converted into double-stranded cDNA. The cDNA can then enter a suitable carrier.

The preferred host-vector system for GM-CSF segregation relies on transcription of the GM-CSF cDNA into the appropriate transformation vector. The appropriate transformation carrier may depend on the temporary introduction of DNA into mammalian cells Parker, Y. Gluzman, T. Maniatis 1981 cell, mammalian cells.V,.Mellon. P) (279-288 27. For the purpose of isolating the required GM-CSF adapters, it is not required that the cells contain exogenous genes that modify the desired GM-CSF output and that exogenous genes can be temporarily introduced into cell types such that these cell types will be replicated (modify The required output over a period of several days. Since the knowable sign is not necessary in the transformation carrier to transfer the DNA and system

Copying According to the present invention, external DNA can be lost as cells grow over a period of 1 to 2 weeks. However, 2-3 days after transferring the appropriate mammary cells, the required products were found to be created and could be identified.

The optimal host-carrier system is based on the development of monkey CV-1 cell lines transformed with a missing replication molecule (Gluzman, Y., 23, 1981, 175-182, Cell). Transformed monkey cells 1-CV contain a deficient SV40-DNA called 1-CV, minus the origin, COS (SV40). They do not contain a complete copy of the SV40 genome but have high levels of the large T antigen and allow replication of SV40 DNA. They also support Efficiently replicates SV40 deleted fragments in the early region. And bacterial plasmids containing SV40 origin of replication (6495-6491 77 Myers, RM & Tjian, R. 1980 PNAS). This system also provides a means to amplify transferred exogenous DNA by replicating the DNA in (induced by) SV40 medium for the purpose of increasing the mRNA level of the protein copied from exogenous DNA. However, other similar systems are also used. Typically, the carriers used for GM-CSF transcription contain various elements such as enhancers, intron promoters, polyadenylation loci, non-coding regions and transport activators as described below.

Pregnant women here can include enhancers. Helpers are functionally different from primers, but they appear to work in association with primers. Their function at the cell level is not understood, but their unique property is the ability to activate or strengthen potentate (transcription) without relying on primers.

Location or union. Primers need to be located upstream of the gene, while auxiliaries can be located upstream or downstream of the primer within the gene in the form of an intron, or downstream of the gene between the gene and the polyadenylation position, or 13 upstream of the polyadenylation position. The inverted primers are functional, but the inverted primers are functional. The auxiliaries work in a cis-acting manner, meaning that they have an effect on the auxiliaries only if they are present on the same DNA strand. For a general discussion of the auxiliaries, see (1983) 314-33.313 Khoury etet al., Cell. The preferred adjuvants for use with mammalian cells have been obtained from animal viruses such as Simian Vinus 40, polyoma virus, bovine papilloma virus, or adenovirus. Typically, the helper should be a virus that is tolerated by the host cell, meaning one that normally infects Hc-type cells. Viral aids can easily be obtained from commonly available viruses. The helper regions of many viruses, such as Rous sarcoma virus and Simian virus 40, are well known. See Luciw et al., Cell 33:705-6 71(1983) (1983) 33:705-716, Cell. As a routine practice in molecular biology, these regions are excised based on published identification maps for the virus in question, and if necessary, multiple locations are made to enable the adjuvant to be delivered to the carrier as required. For example, see Kaufman and colleagues, Journal of Molecular Biology 159:601-621 (1982) 159:601-621,.Mol. Cell Biol. 2(11): 1304-1319 (1982) et al, J. Mol. Biol

(1982). Alternatively the helper can be created from sequence details - the sizes of the viral helper (generally less than about 150 bp) are small enough that this can be done practically.

Another element that must be present in the carrier assembly is the polyadenylation site. This is a DNA sequence located downward from the transcribed regions of the gene - down a short distance where the imitation stops and adenine ribonucleotides are added to form a polyadenine nucleotide tail at the 13th end of the message carrier RNA, and the addition of polyadenylation is important in stabilizing the RNA message carrier. Against degradation in the cell, an event reduces the level of the carrier RNA and thus the level of the resulting protein.

The locations of the addition of polyadenylation in eukaryotes are known. There is a concensus sequence on the genes in eukaryotes: hexanucleotide 51-AAUAAA-31 hexanucleotide, located 11-30 nucleotides away from the point where polyadenylation begins. DNA sequences containing polyadenylation sites can be obtained from viruses according to published reports. Polyadenylation sequences can be obtained from mouse beta-globin and viral 40 late or advanced gene regions, but viral polyadenylation sites are preferred. Since these sequences are known, they can be created externally and linked to carriers appropriately.

The sequence that separates the polyadenylation site from the transfer stop codon is preferably an untranslated DNA sequence, such as an untranslated eukaryotic gene. Because these sequences and genes are not equipped with a primer, they are not copied. The relay must extend a known distance of up to

About 1,000 bases from the stop codon to the polyadenylation site. Generally, the non-transcribed 3′ sequence increases the yield of the product. The carrier can end from about 30 bp downstream of the polyadenylation sequence, but it is preferable to retain the 13′ sequence downstream of the site. Position of polyadenylation in the wild-type environment. Typically, these sequences extend 200 to 600 base pairs downstream from the polyadenylation site.

The presence of introns in the non-transferable part of the carrier can increase the productivity of the product. These introns can be obtained from sources other than cell lines or gene sources. For example, hybrid introns containing splice position 15 of the second intron in the adenovirus tripartite leader and splice position 13 of the immunoglobulin gene that is inserted downstream of the mimicry start position in the adenovirus major latepromoter lead to increased product yield.

In a preferred substance for the production of GM-CSF and the carrier of transcription, there is a gene that activates the transport. Transport activators are genes that encode the specific protein or RNA products that transfer the desired mRNA. The best example is the adenovirus-related gene VAL (VAL), which is a short RNA mimic that overlaps (interacts) with sequences in the nontranscribed region 5 in Late mRNAs (31,543 Thimmappaya et al., 1982 Cell), sequences that are necessary to activate transposition. By VA RNA is located within the mRNA tripartite leaderadenovirus late and connects the adenovirus tripartite leader together from non-contiguous regions in the adenovirus genome and is located at the 5′ end of large late adenovirus copies. It can interfere with (interact) with VA RNA to activate the transfer of mRNA, which...

It contains a tripartite leader sequence. The preferred and replication-specific cDNA union also contains the tripartite leader and VA RNA adenovirus genes.

These mounts can be created by methods well known to those skilled in

Art Pregnancy components such as auxiliaries, primers and the like can be obtained from natural sources or created as described above. Essentially, if components are present in DNA in large quantities, such as components such as viral functions, or if they can be synthesized, such as polyadenylation sites, then with the appropriate use of specification enzymes, large quantities of the carrier can be obtained simply by culturing the source microorganism and digesting the DNA. Using an appropriate endonuclease, the DNA fragments are separated, the DNA containing the desired element is identified, and the process is repeated. The transformation carrier is usually copied in a small amount and then linked to a suitable recombinant carrier automatically (on its own) such as a prokaryotic plasmid or a phage. The pBR322 plasmid can be used in most cases, see Kaufman et al.oP. cit.

Recombinants are used to combine appropriately ligated transposases, such as by transferring a microorganism with a primary nucleus, multiplying the recombinant to a high number, and resynthesising by cell lysis and separating the recombinase from cell debris.

The carriers containing cDNA prepared from the cell that has GM-CSF activity are transferred to E. coli and placed on slides on Petri dishes at a rate of 2000 colonies per dish. The colonies are left on a nitrocellulose filter and the filter is transferred to a new slide that is kept as the master. And after

By cultivating these colonies, offspring are made and carefully joined to the original ones, so that parts of the reproductive filters are identified by the corresponding part on the main slide.

Each propagation filter is cut into portions containing a previously determined number of colonies per portion, preferably about 200-500 colonies per portion. The colonies are cleared from each portion in a medium such as L-Broth soup, the bacteria are collected by centrifugation, and the plasmid DNA is separated. The plasmid DNA from each fragment is then transferred into a suitable host to copy the protein. The preferred recombination carrier here is a strain of the pBR322 plasmid in E. coli from which sequences that are harmful to the eukaryotic cell have been deleted. See Kaufman et al., op. cit, the use of this strain demonstrates no need to delete plasmid remnants before transfer, and after culturing the transferred cells, the medium is tested for CSF activity. A positive test indicates that the colony containing CSF/cDNA is on a specific portion of the filter.

To determine which electrode on the original main filter portion contains GM-CSF/cDNA. Each electrode on the filter part captures and grows. Then the farms are placed in the matrix. Patches are prepared from a horizontal row and a vertical column of a matrix. DNA samples are prepared from each pooled culture and transferred to the host cells for transcription. Floating material from these spots was examined for GM-CSF activity. One vertical column spot and one horizontal row spots should have GM-CSF activity, the common pole of these spots will contain GM-CSF/cDNA, and if the matrix contains more than one positive pole, there will be more than one positive row column. In this case it is necessary to further examine a small number of electrodes.

GM-CSF/cDNA is excised from the electrodes by specific enzymes and can be traced by known methods. It can easily be appreciated that the method described here can be used to obtain GM-CSF from any source. The complete DNA sequence of GM-CSF/cDNA according to the invention is shown in Figure 1 along with the expected amino acid sequence of the transferred GM-CSF protein product. The DNA sequence that encodes the protein that has GM-CSF activity can be modified (developed) according to the present invention, such as shown in Figure 1 by appropriate methods to be functionally equivalent variations in the final GM-CSF protein in which one or more amino acids have been added, replaced or removed without affecting GM-CSF activity. The first (human) bone marrow experiment described here . Also, for example, 1, 2, 3, 4 or 5 amino acids can be replaced by other amino acids. Belgian Patent No. 898,016, included here with permission, describes one exemplary method for replacing cysteine with, for example, serine.

The GM-CSF/cDNA according to this invention includes a GM-CSF/cDNA gene preceded by an ATG codon and a GM-CSF/cDNA coding for homologous species of the GM-CSF protein. One allele pair is shown in full in Figure 1, another we discovered. It has a thymidine residue at position 365 instead of the thymidine moiety shown in Figure 1. The GM-CSF protein of this invention includes a 1-methionine derivative of the GM-CSF protein (Met-CSF) and homologous species of the GM-CSF protein. protein

Mature GM-CSF sequentially in Figure A begins with the sequence ****Ala* Ala* Ala. Its beginning is indicated by the arrow after nucleotide number 59 in Figure 1. GM-CSF will begin with the sequence ****Met* Ala* Pro* Arg, and the homologous species shown in full in Figure 1 have amino acid moiety number 100 (starting with Ala after the arrow).

It may be referred to as CSF-Thr. Another type has a lle notch at position 100 and may be referred to as CSF-lle. The GM-CSF protein of the present invention has a specific activity of at least 710 U/mg of protein and preferably at least 4 x 710 U/mg when assayed with human bone marrow cells.

Host-bearing systems for GM-CSF transcripts can be pronuclear or eukaryotic, but a mixture of GM-CSF is the preferred mammalian transcription system. Transcription is easily accomplished by transfecting pronucleated or eukaryotic cells with GM-CSF carrier.

The DNA sequence obtained by the method described above can be copied directly into mammalian cells under the control of a suitable primer. Non-homologous primers well known to those skilled in the art may be used, and in order for GM-CSF to be transcribed into prokaryotic or yeast cells the guide sequence (or secreted sequence) must be extracted. The position of the coupon at the N-terminus of the GM-CSF protein is shown in Figure 1. This can be done using typical methods known to those skilled in the art. Once GM-CSF is obtained, such as introducing a suitable carrier and transferring the carrier into a suitable host cell, selecting the transformed cells and culturing these transformants to work to modify the activity of GM-CSF. Suitable host cells include such as E coli, yeast, bacteria such as CHO, and insect cells. Also, the GM-CSF protein formed may have a methionine group at the N-terminus of the protein (here it is called Met-CSF). The mature protein formed by pronucleated and eukaryotic cells will be identical in amino acid sequence but the eukaryotic product may be glycosylated to the same or different extent as the natural product. There are different ways to obtain GM-CSF protein

According to the invention shown in examples below. Other methods or materials such as tripods will be readily apparent to those skilled in the art on the basis of the following examples and descriptions.

The transcribed GM-CSF protein can be reactivated in cells with pronuclei or eukaryotes by selection and separation methods known to those skilled in the art. However, a preferred purification process has also been discovered that enables GM-CSF protein from both genetic, cell-climatic, and natural sources to be obtained at a high degree of purity and activity, which is described below.

Summary of preferred purification process:

The present invention covers the problems of the prior art and provides a method for purifying a protein having GM-CSF activity. The GM-CSF protein, according to the present invention, has a specific activity of at least 1 × 710 units/mg of protein, and preferably 2 × 710 units/mg of protein, when examined in a human bone marrow experiment.

According to the present invention, a method for purifying GM-CSF protein involves precipitating the protein with ammonium sulfate at a saturation point of 80% to form pellets containing GM-CSF protein, re-suspending the pellets in a neutral solution at a pH of about 6 to about 8. Using a neutral solution containing GM-CSF on a chromatographic column, emulsifying it with a neutral solution containing sodium chloride, collecting the parts that have GM-CSF activity, collecting the active parts, using them on a C4 reverse phase column.

And emulsification by acetonitrile. Zero effort - 90% to collect the active part.

Brief description of the fees related to the purification process:

Figure 5 shows SDS-PAGE analysis of purified CSF protein.

Detailed description of the purification process:

To purify the GM-CSF protein according to the process of the invention - it can be derived from any of the natural sources described above as primary sources for the process of artificially introducing DNA into the cell in order to change the genetic and climate type of the cell and wrap it along the natural DNA. Such as Mo cell line or UCD MLA-144 Gibbon cell line.

Alternatively, the GM-CSF protein can be formed using methods of introducing DNA into the cell in order to change the genetic type and climate on the cell and wrap itself along the natural DNA - of the invention.

GM-CSFs can be derived from any source of the present invention. Preferably, the appropriate (conditioned) medium from any GM-CSF source is concentrated by ultrafiltration to a protein concentration of at least 0.1 mg protein/ml and then sedimented.

Protein is added by adding ammonium sulfate to 80% saturation. The resulting pellet is resuspended in a neutral aqueous solution at a pH of about 6 to 8. Examples of suitable neutralizing solutions include HEPES, Tris-Hcl, sodium citrate, and the like.

The neutral solution is made functional by column chromatography.

Materials suitable for use in column chromatography are octyl sepharose DEAE, octyisephrose trogel AcA-44, ultrogel trogel and the like.

One or more of these materials can be used in succession to obtain a higher degree of purity.

Functions from each column were collected and examined for GM-CSF activity. The active fractions were collected and diluted with trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA preferably) or the like, and used on the C4 reverse layer column. The CSF activity was then emulsified using 0-90% acetonitrile in TFA or HFBA, preferably at a concentration of 0.10% or 0.15% (v/v), respectively, depending on the acid used to collect the fractions collected on the column.

Fractions with GM-CSF activity were analyzed by electrophoresis with SDS polyacrylamide gel (13.5% gel) as described by (1970) 227.680 Lammi, U Nature. Additional treatments using the aforementioned chromatographic column materials can further purify the GM protein. -CSF for homeostasis gene.

GM-CSF protein fractionated by SDS PAGE gave a heterogeneous GM-CSF protein with an apparent molecular weight in the range of about 15,000 to about 26,000 daltons. This apparent size heterogeneity results from extensive glycosylation of the protein, which is a general characteristic of glycoprotein. Fractionation of less pure samples of Mo cell conditioned medium by SDS PAGE (under non-reducing conditions) and emulsified protein gelation assay yielded

A second protein with CSF activity has an apparent molecular weight of about 28,000 to

GM-CSF activity combines and emulsifies octylsepharose DEAE, trogylated octylsepharose and reverse layer column C4. Unite

Randomly 60% of GM-CSF activity with Con-A sepharose (40% flow through) and can be elicited by methylmannoride.

methylmannoside.

Analysis of the molecular weight of the CSF that is introduced into the cell in order to change the genetic and climate type on the cell - by generation filtration at a low salt concentration or about 30% of the activity gave it an emulsification with a calculated molecular weight of about 19,000, but 70% of the substance behaves in the form of dimmers, It emulsifies at the position corresponding to a molecular weight of 38,000. If 1 mole sodium chloride is included in this column, all the activity will be emulsified in...

If you include 1 mol sodium chloride in this column, all the activity is emulsified in

Broad peak at about 19,000 daltons.

Purified GM-CSF is stable for at least 16 hours when incubated at 4 M (pH 7.4) in 4 M guanidine HCl.

guanidine hydrochloroide in 1 mM, EDTA, 10 mM 2-mercaptoethanol in 30% (vol/vol) ethanol. CSF activity is also constant in 0.1% trifluoroacetic acid (TFA) (pH 2) and 0.1 TFA + acetonitrile 25% (v/v).

As previously mentioned, the GM-CSF protein according to the present invention is described for use in treating myelo-suppression such as granulocytopenia (symptomatic), for example due to chemotherapeutical or radiation therapy for cancer. In addition, special GM-CSF proteins. It is prescribed for use in treating infections (severe injuries). For this use, as indicated, the typical doses prescribed are approximately 200 to 1000

micrograms/patient. It is preferable to inject the GM-CSF protein into the patient's vein in a suitable pharmacy holder. Examples of these holders include a pharmaceutical salt solution and human serum in a salt solution.

In addition, the GM-CSF proteins of the invention have other activities and uses. For example, it has been shown that murine GM-CSFs activate neutrophil white blood cells. It is also expected that the first GM-CSFs of the present invention will also activate the formula's neutrophil white blood cells, so the physiological functions of GM-CSFs are doubled. In the bone marrow, this lymphokine can stimulate the proliferation and diversity of cells affecting the host's immunity, while in the blood periphery, new and existing cells can be activated in a specific immune response. GM-CSF can preserve the neutrophil white blood cells of the formula that circulate in the blood at or far away. About areas of inflammation. Uncontrolled selection and/or activation of neutrophil white blood cells can occur in the pathophysiology of a type of immune system-induced disorder such as rheumatoid arthritis.

Moreover, the invention is understood by reference to the following illustrative contents, which are representative only and should not be taken as specifying the true framework of the invention as described in the elements of protection.

In the examples, unless specified otherwise, the temperatures are expressed as m.

Use endonucleotase selection under conditions and in the manner described by commercial suppliers. The binding reactions take place as described by 245-6 Maniatis et al, supra at, the invention listed here, with permission, by using buffar as described in recipe 246 therein, and using a DNA concentration of 1 - 100 micrograms/ml at a temperature of 23°C for For DNA

The end width is 16 m for DNA with an organic end. Electrophoretic separation was carried out in a 0.5-1.5% agarose gel containing 90 mmol trisborate and 10 mmol EDTA. All radiolabeled DNA is loaded with 23p, regardless of the type (method) of loading used.

Rapid prep is intended for small-scale production of bacteriophage or plasmid DNA as described by Maniatis et- Al-, supra, at p. 365-373.

Example A

Step 1: Mo cell line cultures.

ATCC CRL 8066 (Mo) cells were grown routinely in Alpha (6% CO) or Iscove's medium (10% CO) containing 20% fetal bovine serum (FCS), 2 mM glutamine, 100 units/ ml streptomycin and 100 micrograms/ml penicillin. Cells should be cultured (under cultured) every 4-5 days. Cells are prepared and seeded in Falcon 75 flasks1-T cups in 100-150 ml of medium at a density of 3-4 x 10 cells/ml. Cells will double at 20% FCS every 4-7 days. The growth rate is not constant, and cells may sometimes appear to stop growing and then return again in spurts. Mo cells can be grown in serum-free medium. Survival is better when cells are not washed when transferred from FCS to serum-free medium. The optimal density in serum-free medium (SF) = 10 x 5 cells/ml. Cells will grow slightly (or at least maintain a constant number) for 3 days in serum-free medium and then fed with 20% FCS for 4 days on

the least . This growth line (3 days SF, 4 days FCS 30%) can be repeated weekly if SF medium is needed without obvious damage to the cells or for several months.

Step 2: Test for GM-CSF activity

1- Bone marrow experiment

Get fresh bone marrow. Break the spicules by withdrawing from the cells of a 20, 22 and then 25 needle. Dilute 1:1 with sterile neutral phosphate buffered saline (PBS) (room temperature) and layer over Ficoll-paque (about 30 as BM-PBS over 6 as Ficoll). Isolate at 1500 rpm for 45 minutes at room temperature. Remove the fat and the PBS layer and isolate them. Pip through a low-density layer cloud, wash 2x with PBS and place the cells in RPMI medium (purchased from G1BCO at 1640 RPMI) HlFCS + 10% (heat-activated FCS) for 3 hours to extract adhered cells.

Medium used in slides (fresh):

FCS 20%

0.3% agar dissolved in water cooled to 40°C.

P/S 1% final concentration 100 units/as streptomycin, 100 micrograms/ml penicillin.

10+M alpha-thioglycerol in 2x Iscoves from 102M Cool the agar to about 40M, mix the other ingredients. Cool in a water bath to about 37-38°C and hold at this temperature.

After 3 hours, pull through the cloud of non-adherent cells.

Isolate and promise. Add 2 x 510 cells/ml from the center of the slide and store in a cold bath with a regulated temperature at 37-38°C. Add samples

(eg medium from transfected cells, often a 10 µl sample) to the first row of microtiter slide vessels in pairs. 50 μl of cell suspension was added to each vessel. An additional 50 μl of cell suspension was added to each bowl in the first row. Mix completely and transfer 50 microliters of solution from the first row to the second row, etc. and continue dilutions of 1:3 on the slide. Wrap the slide in film on the outside, incubate 10 - 14 days in 10% carbon dioxide, 37°C in a completely dried cover and observe the colonies.

To monitor the colonies, meaning the total number of colonies growing in each pot in each experiment, several pots are placed on slides without a sample (mock) to obtain a colony number (background). The average number of colonies growing in the mock vessels is subtracted from the number of colonies present in each of the vessels containing the samples. The GM-CSF unit is the amount that stimulates the formation of one colony above the basal level for every 10 human bone marrow cells (placed on slides at a rate of 10/as), then the GM-CSF concentration is below the point of saturation. The sub-saturating concentration is determined by diluting and comparing colonies at different concentrations to find the exact concentration that is below the saturation level.

For this experiment, colonies that contain white blood cell granulocytes, monocytes, or both types of cells are counted, and the cell types in these colonies are determined by capturing individual colonies and cell types.

2- KG-l cell experiment

1-KG cell development (Blood - Part 56, No. 3 (1980) Cell (Blood, Vol

(1980) 3 No) in FCS + Iscoves medium 10% passage × 2 per week and seeded for each passage at a rate of 2 × 10 cells/ml). The cells were used for the experiment between passages

30 - 35. The experiment was exactly as in the bone marrow experiment that was described. They were placed on slides in an agar mixture at a rate of 4 x 10 KG-1 above. As for cells/cells, the number of colonies growing in each vessel is determined and the basic number is subtracted as in the described bone marrow experiment. Above you know that the half-maximum CSF/ml concentration of KG-1 GMCSF unit stimulates it to grow. The KG-1 limit for the number (saturation) of colonies is obtained. Maximum number containing GM-CSF saturation level in multiple vessels

Step 3: Install (configure) holder (B) p90123

Cell Biol. 2 by -130:(11 pAdD26SvpA(3) Description of the transition carrier 1982] 1319. It had the structure shown in Figure 2 [Kaufman at al. Mol. Dihydrofolate reductase plasmid Briefly, this plasmid, which is under cDNA control, contains the mouse dihydrofolate reductase (DHFR) gene and the splice site for Promoteradenovirus 2 (Ad2) major late transporter and splice site 3 derived from adenovirus Adenovirus DNA is present in the latepromoter Ad2major immunoglobulin is present between the immunoglobulin gene. Ammonium globulin present SV40 and early polyadenylation site DHFR and codon sequence 3) derived from) pAdD26SvpA fragment. DHFR coding downstream Maniatis, T. 1981. Cell 27:279-288) pBVOd (mellon, p. Parker, V., Gluzman Y. is known to inhibit replication of pBR322 and does not contain the and sequences. 1981, Nature (London) 293:70-81 (Lusky, M., and Botchan. M. mammalian cells as shown pCVSVL2-TPL into plasmid pAdD26vpA(3) transforming pAdD26SvpA(3) (d) Transforming into plasmid pAdD26SvpA(3) in Figure 2

This is done by digestion. pAdD26SvpA in Pstl by deleting one or two positions using decreased enzyme activity so that partial Pstl is obtained by

(on subpopulation of linear plasmids in which only one Pstl locus is cleaved), then treatment with Klenow, ligation to recirculate the plasmid, transformation of E.coli and screening for deletion of the Pstl locus located at position 13 of the SV40 polyadenylation sequence.

The adenovirus tripartite leader genes and virus-associated genes (VA genes) were inserted into pAdD26vpA(3) (d) as shown in Figure 2 or

pAdD26SvpA(3) (d) is not ubiquitinated by Pvull to make an open linear recombination

Within fragment 3 the first three elements containing the tripartite leader are then digested (851 16 pJAW 43 (Zain et. Al., 1979), Cell) with 1 Xho treated with klenow and also digested with Pvull and a 140 base pair fragment containing the second And part of the third leaders by electrophoresis on acrylamide gel (6% in Tris borate [1982]. al supra) Maniatis et , Tris borate, the 140 bp fragment was ligated to (pAdD26SvpA(3) (d) digested by Pvull , use the result of the link to convert E. coli to tetracycline assay, and the colonies were examined using the Grunstein-Hogness method using a hybridization bar loaded with a 32p to a 140 base pair fragment. DNA was prepared from hybridization-positive colonies to test whether the reconstituted Pvull position 5 or 3 in the DNA 140 base pairs was specific to adenovirus late leaders II and III. And in the correct orientation in the Pvull position on side 5 in the padding of 140 base pairs. This plasmid is called pTPL in Figure 2.

The Ava IID fragment in SV40 containing the SV40 helper sequence was obtained by digesting SV40 DNA with AvaII, exposing the ends with a klenow fragment in 1 Pol, attaching Xhol ligands to the fragments, digesting with Xhol to open the Xhol position and separating the bulk (D) by

Gel electrophoresis This fragment was ligated with cut pTPL 1 Xho to produce plasmid pCVSVL2-TPL. The orientation of the SV40 D fragment in pCVSVL2-TPL was such that the SV40 late promoter was in the same direction as the adenovirus late promoter.

To introduce adenovirus (VA)-associated genes into TPL-pCASVL2 or neither, plasmid pBR322 containing the Hind III B type fragment was constructed into adenovirus, and the B fragment was separated after gel electrophoresis. This fragment is then inserted into pBR322 that was previously digested by HindIII. After turning E. coli for ampicillin resistance. Inserted materials are screened for the insertion of the Hind III B fragment and the direction in which it is inserted is determined by digestion of the pBR322 selection enzyme Ad Hind III B contains the adenovirus type 2 Hind IIIB fragment in the expected orientation in Figure 3.

As shown in Figure 3, the VA genes are obtained from plasmid pBR322-Ad Hind III B by digestion with EcoRI and reactivation of the 1.4kb fragment. The broad-ended fragment was then ligated to the EcoRI position in pTPL (previously digested by EcoRI) after transformation of 101 E. coli HB and testing for tetracycline comparison, and then the colonies were screened by hybridizing the filter with a DNA rod specific for the VA gene. DNA is prepared from hybrid positive electrodes and characterized by specific endonuclease digestion. The resulting plasmid is called...

p91023

2 EcoRI is extracted at p91023. p91023 is cut to the end by EcoRI, generating two DNA parts, one about 7kb and the other about 1.3kb containing VA genes. The ends of both parts are filled in using a klenow part in Poll and then both 1.3kb and 7kb parts are rejoined together. Plasmid (p91023(A

It contains the VA genes and is similar to Grunstein-Hogness by gene fragment

VA and by analyzing the appropriate selection position.

The single Pstl locus in p91023(A) is then excised and replaced by the EcoRl locus. p91023(A) is cut to the end by Pail and then processed by a klenow fragment in Poll to form flat ends. EcoRI ligands are attached to the transverse Patl locus in p91023(A). Linear p91023 (A), with EcoRI linkers attached at the Patl position, is separated from the unbound linkers, digested to the end by EcoRI, and then religated. Plasmid (p91023 (B) is reactivated and identified to have a similar structure to p91023 (A) but the EcoRI position is located at Previous Pstl position.

Step 4: Prepare cDNA Library

Mo cells were stimulated for 16-20 hours with PHA and PMA to help produce their own lymphokine. Cells were seeded at 5 × 10 cells/like in Iscove's medium with 20% FCS, 0.3% (v/v) PHA and 5 ng/ml TPA. Cells were collected by centrifugation. The resulting cells were re-centrifuged in 20 ml of ice-cold low-osmosis buffer (RSB: 0.01 M Tris hydrochloride, pH 7.4, 0.01 M potassium chloride, 0.0015 Mg chloride, 1 μg/ml cycloheximide). 50 units/as RNAsin and 5 mM cithiothreitol. Cells were allowed to freeze on ice for 5 min and then mechanically blown apart by 10 shocks of a well-mounted glass homogenizer recentlyrifuaged at low speed. The homogenized liquid was centrifuged at low speed (2000 RPM in a Beckman centrifuge

Beckman j6) to extract nuclei and non-lysed cells. The floater was suspended on ice while the nuclear pellet was re-equipped at 10 RSB and re-centrifuged at low speed. Collect this second supernatant with the first and centrifuge the combined supernatants at low speed to remove remaining contamination with nuclei and unlysed cells. Bring the supernatant from this to 0.15 potassium chloride by adding 2 potassium chloride and then centrifuging at high speed (25,000 RPM, centrifuge Central SW 28 rotor for 30 minutes) to form a ball on the membranes (to ball the membranes). The membrane pellet was gently washed with cold RSB and then resuspended in 2. An example of RSB containing 2 sucrose and 0.15 potassium chloride. Then two continuous voltages were prepared in SW41 Beckman centrifuge tubes by layering 6 ml of the membrane solution in 2. Sucrose over 2 times RSB with 2.5 sucrose and 0.15 potassium chloride. The tubes were filled to the brim (top) by layering with 2.5 volumes of RSB containing 1.3 sucrose 0.15 and potassium chloride. These gradients were cycled for 4 hours at 27,000 RPM (Beckman, SW41 rotary device) at 4°C. The layer of membrane (between 3 and 1.3 moles of sucrose) was gently removed from the face using an 18 gauge needle and syringe. The membrane fractions were collected from 2 gradients and diluted with 1 volume of distilled water, then brought to Triton X-100 0.5% and sodium deoxycholate 0.5%, then extracted with an equal volume of phenol. The aqueous layer was re-extracted with a 1:1 thread of phenol, chloroform, and finally an equal volume of chloroform. Finally, RNA bound to the membrane was precipitated by adding sodium chloride up to 0.25 F

205 A volume of cold ethanol and incubate overnight at -20°C. The precipitated RNA was collected by centrifugation (4000 RPM for 10 minutes, in a Beckman J-6 centrifuge) and resuspended in 1 volume of distilled water) and from 2 × 910 cells, approximately 1 mg RNA was obtained.

Message-carrying RNA (mRNA) was isolated from total RNA by chromatography methods over 0.5 methylation of dT oligomers. Cellulose, in short, heat the RNA to 570°C for 5 minutes. Shake quickly over ice and then dilute 5-fold by combining with a buffer at room temperature. (0.5 mol LiCl, 0.01 Tris hydrochloride, pH 7.4, 0.002 EDEA and 0.1% SDS). mRNA was precipitated by adding up to 0.25 mol NaCl and 2.5 vol of etlranol ethanol and incubating overnight at -20°C. Collect the precipitated mRNA by centrifugation (30,000 RPM for 30 minutes in a Beckman SW55 rotor). The tube was carefully decanted and the mRNA pellet was resuspended in 50 ml of water. The reconverted mRNA was resuspended in 0.25 sodium chloride and extracted once with a 1:1 mixture of phenol and chloroform and extracted three times with chloroform. mRNA was precipitated by adding 2.5 volumes of ethanol. The mixture was frozen and spread several times in a dry ice/ethanol bath and then centrifuged for 15 minutes in an Eppendorf centrifuge. The tube was carefully decanted and the mRNA pellet was resuspended in 20 μl of distilled water. The final yield was approximately 30 μg of mRNA.

The first strip was prepared using typical methods. Briefly, dilute 10 μg of membrane-specific mRNA in 100 μl of cDNA synthesis reaction mixture containing 300 mM Tris, pH 9.4. 140 1 mM potassium chloride, 10 mM magnesium chloride MgC12, 10 mM mercaptoethanol, 500 μM each of dCTP, dGTP, daft, and dTTP, 5 μg of dT oligo (phosphorylated and medium size 12-18) as primer, 150 uCi of 32p dCTP (400 Ci/mmnol) and 20 units of RNAsin ribonuclease inhibitor. The reaction was started by adding 100 units. of reverse transformation enzyme and incubation for 30 minutes at 42°C. Stop the reaction by adding up to 40 mmol EDTA and analyze the RNA by incubating for 20 minutes at 65°C in 0.2 mol sodium hydroxide.

sodium hydroxide.

The base was neutralized by adding 20 μl of 2 mol Tris, pH 7.4. Then the reaction mixture was extracted with phenol/chloroform, and extracted again with 50 μl of 10 mM Tris, pH 7.5, 1 mM EDTA, and the aqueous bases were collected. Convert the first strand of cDNA into double-stranded cDNA by incubating for 12 hours at 16°C with 40 units of the klenow fragment of DNA polymerase 1 in 100 μl of a reaction containing 50 mM potassium phosphate, pH 7.4, 2.3 mM. DTT, 2-mercaptoethanol, 10 mM magnesium chloride, 150 μM each of 4-deoxyribonucleotide triphosphate and 25 μCi of 32p dCPT. The reaction was stopped faithfully by phenol/chloroform

chloroform and extract the unincorporated third phosphate by passing over 1 instance of the G-50 sephadex column. The removed parts were collected and ethanol was precipitated.

The cDNA pellet was washed with cold ethanol and then resuspended in 200 μl ul of 20 mM Tris, pH 8, 1 mM EDTA, 80 μM s-adenosyl-methionine, and 300 units of EcoRI methylase. Methylated for 30 minutes at 537°C. The reaction was stopped by extraction with phenol/chloroform and the methylated cDNA was collected by ethanol precipitation.

The cDNA pellet was rinsed with 70% ethanol, then resuspended in 200 μl of Buffer Maniatis (S1 and colleagues) and incubated with 200 units of S1 nuclease at 30°C for 30 minutes. The reaction was stopped by extraction with phenol/chloroform and the cDNA was collected by ethanol.

Double-stranded cDNA was exposed by incubation in 100 μl of Tris buffer, pH 7.4, 50 mM sodium chloride, 10 mM 2-mercaptoethanol, and 500 μM of each of the four third-order deoxyribonucleotides. Deoxynucleotide triphosphates with 25 units of Kienow at room temperature for 30 minutes. The reaction was stopped by extraction with phenol/chloroform and the cDNA was collected by ethanol precipitation.

Ligation of cDNA in 50 μl of T4 ligase (Maniatis and colleagues) with 500 pMoles of Rl ligases purchased from England Laboratories (sequence: pCGGAATTCCG) using 2000 units of T4 ligase length

Night at 16pm. The reaction was stopped by incubation at 70°C for 20 minutes and then diluted to 300 μl so that the final concentration of the salt was 0.1 M NaCl, 10 mM MgCl, 50 mM

Tris chloride, pH 7.4. The cDNA was then digested for 2 minutes at 37 mM with 700 units of EcoRl. The reaction was stopped by extraction with phenol/chloroform and the cDNA was collected by ethanol precipitation. The grape was resuspended in 50 μl of TE and passed over 5 liters of Cl-48 column. The retracted parts were collected and precipitated with ethanol. Electrophoretic separation of the precipitated cDNA was carried out through a 1% agarose gel in Tris acetate buffer in the presence of 1 μg/ml ethidium bromide. Ethidium bromide cDNA was separated at a size range of 500-4000 base pairs by generation using the typical glass powder method. The emulsified cDNA was extracted by

Phenol/chloroform, precipitated with ethanol and the pellet reconstituted into a suspension

(after rinsing with ethanol) in 50 μl of TE. The final yield was 100–500 ng of cDNA.

Preparation of the transcription carrier (p91023 (B) described above. Ligation of the EcoRl-digested and phosphatase-treated carrier (500 ng) with 100 ng of cDNA in 100 μl of reaction (typical T4 ligase reaction) overnight at 16°C. Stop the reaction by extraction. by phenol/chloroform and then collected the bound cDNA - by ethanol precipitation after adding 5 μg of tRNA as carrier.

The ethanol-precipitated DNA was rinsed with 70% ethanol and then resuspended in 100 μl of TE. This DNA was used in amounts of 4 μl of transform E.coli MCI016 (4 μl in 100 μl transform).

Each of the 25 transformants was spread (spread) on a 150 mm Petri dish with 1% agar, L-soup, 10 μg/as tetracycline (Tet slide) and incubated overnight at 37°C. Approximately 2,000 colonies grew on each slide, making a total of 50,000 colonies. After reaching approximately 0.5 mm in diameter, I transferred the colonies to nitrocellulose disks (137 mm) by carefully placing a dry filter on the surface of the slide and then gently peeling the filter off. All colonies on the slide were transferred to the filter which was then placed (colony side up) over a fresh Tet slide. After allowing the colonies to grow for several hours. Then prepare one strain from each filter. Place a fresh moisturizing filter exactly on top of the original filter, pressing them together, peeling them off, then returning each filter to a fresh Tet slide and incubating the slides overnight at 37°C. Each lineage (offspring) was carefully marked so that it matched the original candidate.

Step 5: Prepare the Plasmid DNA Pool

Carefully divide each of the 25 reproductive filters into eighths using scissors and trace the direction of each eighth relative to the original main filter. Colonies were wiped from each portion in 10 ml of L-Broth broth. Bacteria were collected by centrifugation (3000 RPM, 10 minutes, Beckman J 6-centrifuge), resuspended in 0.6 ml of 25% sucrose, 50 M Tris-HCl, pH 8, and transferred to Primary cells (protoplasts) by adding 0.12 ml of lysozyme 5 mg/ml and incubating on ice for 5-10 minutes. The protoplasts were again incubated at chamber temperature for minutes after adding 0.125 ml of 0.5 M EDTA and then lysed by adding 0.12 ml of 10% SDS in 50 mM EDTA.

mM Tris-HCl, pH 0.8 Gently mix the lysate, incubate at room temperature for 15 minutes, then precipitate the protein and chromosomal DNA by adding 0.3 ml of 5 M sodium chloride.

After incubation on ice for 15 minutes, the analyte was centrifuged in a cold Eppendorf centrifuge for 30 minutes. Carefully pick off the floater behind the sticky DNA/protein globule and dilute by adding 2.5 volumes of water. The mixture was extracted with 1 M of phenol, the layers were separated by centrifugation (10 K for 10 minutes in a 34-Sorvall SS rotary device) and the aqueous layer was extracted into a fresh tube. The DNA was precipitated by adding 0.5 M of 5 M sodium chloride and 7.5 M of cold ethanol and freeze the mixture several times in a dry ice ethanol bath. The precipitate was collected by centrifugation (10 K for 15 minutes in 34-Sorvall SS), re-suspended in 0.3 ml of 0.3 M sodium acdtate and re-precipitated ( In a tube Eppendroftube) by adding 1 D of ethanol. After 10-15 minutes in a dry ice ethanol bath, the precipitated DNA was collected by centrifugation (5 minutes, in an Eppendrot apparatus) and the pellet was resuspended in 100 μl of sterile TE (10 mM Tris, pH 8, 1 mM EDTA) and 5-10 micrograms of plasmid DNA were obtained from a typical preparation. Each preparation contained DNA from 200-500 colonies on the original filter, and a total number of 200 DNA samples were prepared from the 25 filters.

Step 6: Isolate (separate) GM-CSF

Each of the DNA samples from step 5 was transfected individually into monkey M6 COS cells as described below.

M6 cells were routinely grown in Eagle's modified medium B

Dlbecco's DME (available from Gibco) contains 10% bovine fetal serum (HIFCS), divided twice a week at a 1:6 dilution. After dividing for approximately 24 hours, M6 1:6 cells are ready for transfer. Approximately 24 hours before transfer, M6 cells were seeded 1.2 x 810 (split 1:6) in a cell plant (available from Nunc) in 1.5 L of DME + 10% HIFCS. Immediately before transfer (immediately before transfer), the slides were aspirated and washed twice with 7 volumes of serum-free SF (DME).

The DNA was dissolved in 0.1 M Tris (pH 7.3) and added to DME medium containing 2 mM glutamine.

100 µg/as streptomycin, 100 units/as penicillin and 0.25 mg/as DEA dextran. Dextran reaches a total volume of 4 times with DNA Tris solution. 4 times of the medium containing dissolved DNA was added to the slide containing M6 COS cells and incubated for 12 hours. After incubation, the cells were rinsed once or twice with 7 i.m. SF DME and then 5 i.m. DME with 10% HIFCS, 100 units/m penicillin, were added.

100 µg/ml streptomycin, 2 mM glutamine, and 0.1 mM chloroquin and the cells were incubated for 2.5 hours.

After 2.5 hours, I rinse once with SF DME and add 10 As + DME HIFCS 10%. Harvest by extracting the conditioned medium after an additional 4-26 hours of incubation (incubation).

The conditioned medium from each transfer was examined for GM-CSF activity using a 1-kg test. For each sample positive for GM-CSF activity, the electrode on the original main filter responsible for GM-CSF activity was identified. For example, a single transfusion positive for GM-CSF activity captured all colonies in the original master filter fraction from which the DNA sample was derived. Approximately 320 of these colonies were picked up in 3 volumes of L-Btoth broth + µg/ml tetracycline, the colonies were grown overnight, and the 320 colonies were placed in an 18 x 8 matrix. Clustering spots were prepared from each horizontal row and vertical column in the matrix (a total number of 36 spots) (note: the last horizontal row has only 14 clones). DNA samples were prepared from each clustering colony and then inquired for COS cell transfer. Floating material from these was examined. Transfers using the 1-KG colony experiment, and 2 positive results were obtained from this group of transfers: one in the vertical column and the other in the horizontal row. The general culture of these spots (clusters) contained a GM-CSF clone electrode.

2 individual clones were isolated from this culture, and miniprep DNA was prepared from 10 ml cultures in L-broth broth as described above. The floating material from these transfers was examined using the 1-kg test in addition to the bone marrow test for GM-CSF. The four clones, each containing 750 base pair fragments, all directed transcription by M6 COS cells that had high levels of GM-CSF activity as determined in one of the two experiments, while the three clones

The other was not directed. Also, the code region of GM-CSF must be located within a padding of 0.75 base pairs.

The DND sequence of the GM-CSF codon was excised from the positive pole shift carrier by EcoRI digestion and followed using typical dideoxy sequencing methods after the underpolarized fragments (combination) in the M13 positions to obtain the sequence shown in Figure 1. The p91023(B)-CSF plasmid that was first shown to direct GM-CSF transcription in COS cells was named pCSF-1, and this plasmid was deposited by the American type culture pool in strain MCI061 in EcoRl under ATCC accession number 34754 on July 2, 1984.

Step 7: Transcription of GM-CSF protein

Monkey M6 COS cells transformed with p91023(B) carrier containing GM-CSF/cDNA were grown as described in step 6 to generate GM-CSF protein in the culture medium.

I mean, I dissolve 1 mg of this (1-pCSF) in 1 ml of 0.1 M Tris, pH 7.3 and add to 600 ml of DE containing 2 mM glutamine, 100 units/ml streptomycin, 100 µg/ml penicillin (P/S) and 0.25 mg/ml DEAE Dextran (molecular weight 500,000 from Pharmacia). 600 ml of DEAE DNA dextran solution was added to M6 COS cells in a cell factory and incubated at 37°C for 2 1 hour. After incubation, the cells were rinsed once with 900 ml SF DME containing 0.1 mM chloroquine (HIFCS), 10% chloroquin, 2 mM glutamine, and P/S (penicillin-streptomycin). After rinsing the containing medium

Chloroquine 3 cells with DME SF and 1500 ml of DME with 10% HIFCS. After 30 hours, the cells were washed with SF DME and the transfected cells were allowed to adapt to the medium for 24 hours at 37°C. Rinse the conditioned medium and replace with another 800 ml of SF-DME. The cells were allowed to adapt to this medium for 24 hours, then the conditioned medium was removed, and as soon as possible after harvesting, the sample of the conditioned media was concentrated 20-fold by over-pressurized filtration using a 2.5 liter Amicon chamber and a membrane (5,000 YM5 MW cutoff).

Step 8: Purify GM-CSF which is introduced into the cell to change the genetic and climate type of the cell.

Bring 200 ml of concentrated conditioned medium (from 4 liters of starting material. Step 7) to ammonium sulfate, 3% saturate by adding solid ammonium sulfate and extract the precipitated protein by centrifugal force. Bring the floater to 80% ammonium sulfate saturation by adding more ammonium sulfate and collecting the precipitated protein by centrifugal force. The pellet was resuspended in 5 ml of 20 mM sodium citrate, pH 6.1, containing 1 M sodium chloride. The dissolved protein was used on a 1.6 x 100 cm column of Ultrogel AcA54, weighed in the same beaver. The GM-CSF activity was emulsified from the column by an apparent molecular weight of 19 kDa or about 90 ml. It was observed that if gel filtration was performed at low ionic strength, the GM-CSF activity was emulsified from the column in two positions - with apparent molecular weights of about

19 (kDa) and 38 kDa, GM-CSF can easily be dimers. I collected the active parts and prepared 15% TFA (by adding 10% TFA) and used it on a Vydac C4 column (0.46 x 25 cm) weighed in 0.1% TFA. The column was supplied with a linear gradient from zero to 90 of acatonitrils (6 ml/min, 340). mCl) in 0.1% TFA. GM-CSF activity emulsified between 39-43% acatonitrils (fractions 16-20). A 20 μl sample from fraction 19 was analyzed by polyacrylamide gel electrophoresis for SDS (13.5% gel as described by Lammli (1970) 227.680 In Nature, a single broad protein epitope observed with an apparent molecular weight of 18-26 kDa and a wider GM-CSF size ratio is a common characteristic of glycoproteins, and is thought to reflect extended and varied addition of carbohydrates. The protein in fraction 19 was subjected to Edman analysis using an Applied Biosystems gas phase microsequenator. From approximately 20 μg of protein used, the sequence of the first 15 amino acids (APARSPSPSTQPWEH) was obtained. The high yield of single protein sequences strongly confirms that the GM-CSF protein in fraction 19 Purified to homogeneity, bioassay indicated that fraction 16 had 3 x 710 adsorption units/A280 adsorption units. Since typical proteins in aqueous solution have an average extinction coefficient of 0.8 to 1.2 A280 adsorption units/mg of protein. The specific activity of purified GM-CSF is between about 1 x 710 and about 4 x 710 U/mg by screening using the human bone marrow test.

Example B

GM-CSF Gibbon Federation

Step 1: Prepare mRNA from gibbon T cells

A sample of the venomous gibbon-specific T cell line 144 UCD-MLAK was cultured for several weeks in 1640 RPMI (purchase from Bibco) and 20% fetal bovine serum (FCS) until a total cell count of 1 × 910 was obtained, and the cells were induced to form levels High levels of CSF by stimulation for several 24 hours in the presence of 10 ng/ml 0-12 tetradecanoyl phorbol 3-acetate (FPA) at 1540 PCS + RPMI 1%. Cells were harvested by centrifugation (1000 rpm, 5 minutes), washed once with phosphate buffered saline (PBS) and finally collected by centrifugation.

Membrane bound polyzyme (MBP) mRNA was prepared from these cells using the same method described in Example A for RNA preparation in Mo cells.

Step 3: Reaction of the second cDNA strand

The first cDNA strand pellet (step 2) was resuspended in 50 ml of water and the synthesis of the second strand was carried out in a typical reaction mixture with E.coli polymerase I ligase E.coli and RNAse H. The reaction was incubated overnight at 16°C, then incubated. For 1 hour at 37°C, the reaction was stopped by adding EDTA and extracted with phenol/chloroform. The cDNA was separated from the unincorporated triphosphate by chromatography on a CL-4B buffer. The separated parts were collected and the cDNA was collected by ethanol precipitation.

Step 4: Preparing cDNA that is introduced into the cell to change the genetic type and climate of the cell and wrap itself along the natural DNA.

The cDNA pellet (step 3) was resuspended in 75 µl of water. Homopolymeric C tails were added to the cDNA ends. Adding 10 µl of cDNA solution to 25 µl of the typical reaction mixture with final transferase and incubating at 30°C for 5 minutes. Stop the reaction. Add EDTA up to 40 mM and stop the activity with heat at 68 C for 10 minutes. 10 ng of cDNA with a G-tail was annealed by 50 ng of pBR322 with a G-tail. Purchase from NEN in 10 μl of Tris 10 mM, pH 7.5, EDTA 1 mM and sodium chloride 100 mM. The agent was annealed for 10 minutes at 68°C, then for 2 hours at 57°C.

Step 5: Bacterial transformation

Strain 1061 MC was grown in Ecoli in L-broth chilled over ice, harvested by centrifugal force and treated with calcium chloride to prepare it for transformation. Incubate 5 μl of the cDNA annealing reaction with 200 μl of bacteria treated with calcium chloride. Performed (15) transformations

Using each annealed cDNA, it was spread over 15 cm slices of 1% agar L broth containing 10 μl/ml tetracycline. Approximately 1,000 colonies grew on top of each slide.

Step 6: Reproduction slices

Each of the 10,000 colonies was picked up from the transformation with forceps 1, transferred to fresh slides (500 per slide in a grid) and grown overnight at 37°C.

Colonies were lifted from each slide by pressing a dry nitrocellulose filter firmly against the surface of the slide. Reproduction filters were prepared from each of these major filters. The master filters were stored at 4°C and the breeding filters were treated with base and returned again to prepare them for hybridization.

Step 7: Prepare hybridization rods loaded with 32p

The cDNA was isolated from 1-pCSF by digestion with an E.coli-specific enzyme and electrophoresis in an agarose gel with Tris acetate and ethidium bromide. The loop containing the cDNA fragment was cut from the gel and purified by the pulverization method

(crushed).

300 ng of the cDNA fragment was then added to 1 μL of 10 × 10 T4 DNA polymerase (0.23 M Tris acetate, pH 7.9, 0.66 M potassium acetate, 0.1 M magnesium acetate, and 10 mM dithiouritol). dithiothreitol) and 3 units of T4 DNA polymerase (England Laboratories) and diluted with water to 10 μl after incubation for 5-10 minutes at 37°C. This strand was combined with 1 μl of 10× T4 DNA polymerase, 1 μl of a 2 mM solution of dGTP, dTTP, dCTP, and 10 μl of 32PdATP (10 units Ci/ui, 3,300 mmol/Ci, and 3 units of Tr DNA polymerase and then incubate the reaction for an additional 20 minutes at 37°C.

Unincorporated triphosphates were separated from the loaded cDNA by chromatography on a Sephadex G100 column. A second rod was prepared from an oligonucleotide, creating the sequence:

ATC TGG CTG CAC AG which is complementary to the amino terminus in the CSF code region. This oligonucleotide is loaded with a 32p dATP at the 5′ end using a typical polynucleotide kinase reaction.

Step 8: Isolate GM-CSF cDNA electrodes

In the same manner as a typical two-hybrid assay, 45 clones were hybridized with the T4-loaded pCSF-1 cDNA, of these approximately 20 also hybridized to the oligonucleoid-loaded rod. The code region in one of these was followed and the sequence details showed a number of base substitutions, some of which resulted in an amino acid difference in the transcribed protein. These differences are shown in Figure 1 above the DNA sequence of the recombinant human GM-CSF gene in Example A.

Example C

GM-CSF association of lymphocyte-specific mRNA in the periphery

Step 1: Prepare mRNA from lymphocytes in the periphery.

Lymphocyte cells were prepared from the periphery of blood from 4 plasmapheresis products (purchased from the Red Crescent) by fractionation over the ficoll Hypaque gradient light intensity in a medium of 1540 RPMl in the presence of fetal calfserum 5% and phytohemmaglutinin 0.17%. And 10 ng/ml phorbal myristate acetate (PMA) at a density of 2 x 610 cells/ml (a total number of 6 x 910 cells was obtained). The cells were harvested by centrifugal force.

(100 rpm/min) washed once with a neutral salt solution (PBS) and finally collected by centrifugation.

Cytoplasmic RNA was prepared by the gentle lysis method in which cells were resuspended in 50 ml of lysis buffer.

Cold Triton (40 mM sodium chloride, 1.5 mM magnesium chloride, 10 mM Tris, pH 8.6, 0.5% Triton Buy from Biotec. Divide this analysis product into two equal parts and place each part in layers on top of a 10 ml pad of lysis buffer containing 20% sucrose. The cell nuclei were extracted by cold centrifugal force (4°C, 400 rpm for 5 minutes). The upper layer (cytoplasmic extract) was gently removed and sodium dodecyl sulfate (SDS) was added to a final concentration of 1%. This solution was extracted twice with an equal volume of phenol chloroform (1:1 strand) and the RNA was precipitated by adding 2.5 volumes of cold ethanol. The precipitated RNA was collected by centrifugation (15 minutes, 4000 rpm) and resuspended in Tris 0.01 M, pH 7.5, EDTA 1 mM, sodium chloride 0.25 M (PFR TE, sodium chloride 0.25 M). It was re-precipitated by adding 2.5 volumes of cold ethanol. Finally, the RNA was collected by centrifugation and resuspended in 5 ml of water, and the final yield was 7.5 mg.

The carrier RNA was isolated from kidney cytoplasmic RNA by selection over oligo dT cellulose. Preheat 2.5 mg

of total RNA to 65°C for 5 minutes. Sodium chloride was added to 2.5 M and the RNA was allowed to cool to chamber temperature. This RNA was passed over a 1 ml column of oligo-dT cellulose weighed in TE + 0.5 M sodium chloride (PFR). Unbound RNA was extracted by washing the column vigorously with a conjugate buffer. Emulsify the drawn carrier RNA with 3 ml of water and precipitate by adding 0.2 ml of 4 M sodium chloride and 2.5 volumes of cold ethanol. Collect the precipitated inRNA by centrifugal force (30 minutes at 25,000 rpm). The final pellet (approximately 100 micrograms) was reconverted.

To be resuspended in 50 µl of water.

Step 2: Reaction of the first cDNA strand

Dilute 20 μg of PBL mRNA in 50 μl of cDNA synthesis reaction containing 100 mM Tris, pH 8.4, 140 mM potassium chloride, 10 mM magnesium chloride, 10 mM 2-mercaptoethanol. 400 micromolar of each dATP, dGTP, dCTP and dTTP, 5 μg of dT oligo (average size 2 1 - 18) as primer, 25 uCi of 32P dCTP (400 Ci/mmole) and 20 units of RNAsin ribonuclease inhibitor. The reaction was started by adding 60 units of reverse conversion enzyme at 37 M . Incubate for 30 minutes at 42°C. The agent was stopped by adding 40 mM EDTA and extracted with an equal volume of phenol saturated with water. The phenol was extracted again with 50 μl of TE buffer, and the aqueous bases were collected. cDNA/RNA hybrids were separated from unfused triphosphates by passing the combined aqueous layer over 5 ml of a CL 4B Sepharose column (purchased from Sigma) equilibrated with TE. I collected the parts that were removed from

The column was brought to 250 mM NaCl and the nucleic acids were precipitated by adding 2.5 volumes of cold ethanol. The hybrids were collected by centrifugation for 30 minutes at 40,000 rpm. The final pellet (2.5 μg of cDNA) was reconstituted in 50 μl of water.

Step 3: Reaction of the second cDNA strand

Creation of the second cDNA strand by the dual action of the enzymes DNA polymerase I in E.coli, DNA ligase in E.coli, and RNAse H in E.coli. The reaction mixture (50 μl) contained 20 mM Tris, pH

8, 4 mM magnesium chloride, 1.2 mM EDTA, 25 μM NAD, 100 μM each of dCTP, dGTP, dATP, and dTTP, and 50 uCi of 32P dCTP (3,000 Ci/mmole). The reaction was carried out by adding 3 units of polymerase. RNA, 0.5 units of DNA ligase and 0.75 units of RNAse H and incubated at 16°C for 18 hours. Then at 37°C for 1 hour, then stopped by adding 40 mM EDTA and extracted with an equal volume of phenol. The phenol layer was re-extracted with 50 μl of TE, the aqueous layers were collected, and the cDNA was separated from the unincorporated triphosphate by chromatography on a CL-4B Sepharose column as described above for the first strand. Depending on the incorporation of 32P, the amounts of the first cDNA strand are transformed into the double-stranded form.

Step 4: Prepare the cDNA that is to be introduced into the cell in order to change the...

Genetic type and climate on the cell and wraps around the normal DNA.

Homopolymeric C tails were added to the ends of cDNA by gently heating 400 ng of cDNA in 50 μl of reaction mixture containing

On 1 nM of 2-mercaptoethanol, 1 mM of CoCl2 and 9 units of terminal deoxynucleotidyl transferase at 30°C for 5 minutes. Stop the reaction by adding 40 mM EDTA and heating to 68°C for 10 minutes. 200 ng of tailed cDNA was annealed by 500 ng of G-tailed 153 PAT (purchased from Amersham) in 100 μl of Tris 10. mM, pH 7.5, 1 mM EDTA, and 100 mM sodium chloride. The annealing reaction was performed at 57°C for 2 hours after a 5-minute previous incubation period held at 68°C.

Step 5: Bacterial transformation

The product of the cDNA annealing reaction was used directly to transform the MC1061 E.coli strain. A fresh colony of bacterial cells was used to inoculate 50 ml of L-Broth broth and grown for several hours until the optical density was 0.25 at 550 nm. The cells were cooled on ice and harvested by centrifugal force (2000 rpm for 10 minutes). The pellet was resuspended in 10 ml of cold 0.1 M calcium chloride and allowed to remain on ice for 10 minutes. The cells were collected by centrifugal force (2000 rpm for minutes) and resuspended in 2.5 ml of 0.1 M calcium chloride. Incubate 10 µl of cDNA anneading reaction with 200 µl of bacteria treated with calcium chloride for 30 minutes on ice, then for 2 minutes at 37°C - followed by the addition of 0.8 ml of L-Borth broth and a final incubation for 30 minutes at 37°C. On average, 1,500 bacterial colonies grew on a slide until the total number reached 30,000 colonies.

Step 6: Reproduction on slides

The original colonies growing on each slide were transferred to 137 mm nitrocellulose filters by pressing a dry filter over the colonies and lifting them off the slide. Two identical strains were prepared from each original candidate using (typical slide propagation methods) in which each original candidate was carefully placed colony-side up on a sterile square of 3 mm Whatman filter paper - mounted on a square piece of glass. Carefully filter a new, previously moistened nitrocellulose filter

Above the main filter and cover with a second sterile square of filter paper

The entire sandwich is then pressed together firmly with a second piece of glass. The compressed filters were given in the form of number sandwiches and 3 holes the size of a pinhead were cut through them asymmetrically so that they would be collected together in the future. The strains were removed from the mother and placed colony-side up on an agar-soup slide containing tetracycline. A second strain was prepared immediately in the same manner. Each master filter was returned to a slide and all slides were incubated at 37°C for hours until bacterial colonies were reduced to approximately 1 mm in diameter. The original parent filters were stored at 4°C and strains were prepared for hybridization as described below.

Step 7: Prepare filters for hybridization

Place each propagation filter (step 6) and colony side up on filter paper (3 mm Whatman) dipped in 0.5 M sodium hydroxide and 1.5 M sodium chloride for 7 minutes. The filters were transferred to neutralizing filter paper, immersed in 1 M Tris, pH 7.5, and 1.5 M sodium chloride for 2 minutes, then transferred to a second neutralizing filter set for 5-10 minutes. Finally put

The filters were immersed in SSC (sodium citrate 0.15 M, sodium chloride 0.15 M, pH 7.4) for 5 minutes and dried with air and again under pressure at 80 C for 1-2 hours.

Step 8: Isolate the GM-CSF electrodes

Filters in pairs were converted into rods by a filler of pCSF-1 cDNA loaded with radioactive material, prepared as described above in Example B, and 20 colonies hybridizing with cDNA were picked up. I picked 12 of these from the main filter and grew them overnight in L-borth broth. For further analysis, the specification enzyme (PstL) digest components of the DNA samples (rapid preparation) from these electrodes indicated that there were 3 near full length. One of these was followed by a sequence The GM-CSF coding region in this clona is identical to the corresponding pCSF sequence but has an F at position 365-CSF-lle.

Example D

Helper transformation and amplification of GM-CSF sequence in CHO cells plasmid Introduce p91023(B)-CSF into DUKX-Bll cells deficient in Cltasin & CHODHFR)

Urlaub PNAS (1980, 4216: 77) through the protoplast union, as described (1981, 752-743 l. Sandri-Goldin et al., Mol. Cell. Bio). The growth and stability (survival) of cells was described (1981, 621-601 150 Kaufman & Sharp, J. Mol. CHO Bio) For single protoplasts, p91023(B)-CSF-l was introduced into E.coli HB101 and the bacteria were grown in 50 ml of M9 salts containing 0.5% casamino acid and 10 μg/ml tetracycline - to an absorbance of 0.9 at 600 nm. Chloroamphenicol was added. Chloramphenical to 250 µg/ml and the culture was incubated at 37°C for an additional 16 hours to increase the number of plasmid copies. The cells were centrifuged at 3,000 x g for

10 minutes at 4 C and suspended in 2.5 ml of cooled 20% sucrose - in 50 mM Tris chloride, pH 8, 0. Add Lysozyme (0.5 ml of a 5 mg/ml solution in 0.25 M Tris chloride, pH 8 ) Hang the mixture over ice for 5 minutes. Add EDTA (1 ml of 0.25 M EDTA, pH 8) for an additional 5 minutes on ice, then slowly add 1 ml of 0.05 M Trischloride, pH 8. Incubate the suspension for 15 minutes at 37°C until the bacteria have grown to Protoplasts. Then the suspension was slowly diluted with 20 ml of preheated medium containing 10% sucrose and 10 mM magnissium chloride and heated at 37 C for 15 minutes. A solution of protoplasts (approximately 910/ml) was added to CHO, DHFR-deficient DUKX-Bl l cells in 6 slices (approximately 1 - 2 x 410 cells/vessel) at a ratio of approximately 1 - 2 x 410 protoplasts/cell and the protoplasts were cultured into the cells by Methods: Centrifugation at a rate of 2000 rpm for 8 minutes in a swinging microtiter dish, model IEC. After centrifugation, the float was removed by vacuum, and an amount of 2 ml of a 50 mM polyethylene glycol solution from (Baker Chem. Co.) PEO-1450 was added in 5 ml of medium - to each bowl of 6

Vessels) The cells were centrifuged again at a rate of 2000 RPM for 90 seconds, the polyethylene glycol solution was extracted, and the slides were rinsed 3 times with 4 ml of medium/vessel. Trypsin was added to cells in 10 ml of media containing 10% fetal bovine serum, and centrifugation was performed in a conical tube at a rate of 500 RPM in a centrifuge.

A clinical. Pellet cells were collected from 3 vessels and placed in a 10 cm tissue culture dish. Fresh medium containing 100 μg/ml kanamycin, thymidine, adenoxine, was added.

Deoxyadenosine, penicillin and streptomycin, and 10% calf serum. He dialyzed each slide. Kanamycin was included to prevent the growth of bacteria that had escaped from turning into protoplasts.

Two days later, the cells were cultured 1:15 in alpha media with 10% bovine fetal serum, dialyzed, penicillin, and streptomycin, but lacking in nucleosides, like cell oil, once fixed using the same optional media (lacking nucleosides) after 4-5 days. days.

Colonies appeared after 10-12 days under culture in selective media, and two linear plans were followed to select and enlarge methotrexate (MTX). In the first layout, individual transformants were isolated, recombinant on the basis of DIR copies, followed by each cione being expanded under conditions to maximize the copy number of foreign DNA in the sense of growth in increasing concentrations of methotrexate. In the second layout, a spot was isolated from several independent transformations based on DHFR transcripts and strung together under conditions to amplify foreign DNA in the sense of growth from increasing concentrations of methotrexate. Individual clones were then isolated from the selected cluster and analyzed for GM-CSF cloning. These slones with high levels of GM-CSF transcripts were grown back under conditions of foreign DNA amplification (i.e. growth in increasing concentrations of methotrexate in the culture media).

In one experiment, seven DHFR mutants were collected in alpha medium deficient in nucleosides. These cells were then grown in increasing concentrations of MTX, starting at 0.02 microM and then serially increasing to 0.1, 0.5, and 2 microM MTX. When examining GM-CSF activity in an experiment

The 1-KG cell produced these cells from 3,000 to 12,000 units/ml. The selection was combined in 0.5 μM MTX and in 2 μM MTX and the clones obtained in 0.5 μM MTX (010, D2, and B6) were then selected for growth in 2 μM MTX, and then screened for GM-CSF activity in an experiment. 1-KG cell, combined cell lines had 15,000 to 30,000 units/unit of GM-CSF activity and the GM-CSF formed according to this example has the amino acid sequence given for the CSF-Try in Figure 1.

Example e

Transcription of GM-CSF in E.coli from the PTALC-185R carrier is given in Figure 6. The codon sequence encoding GM-CSF begins with the epitaxy sequence ATG CCA CCA CCT CCT TCT CCA TCT CCA TCT ACT, which identifies eleven amino acid moieties. The first is in exuded GM-CSF, and the rest of the sequence that carries the GM-CSF code in PTALC-l85R is identical to its counterpart in 1-Pcsf. Nucleotides 96 - 447 are followed by the sequence TTA TAA TAG, and immediately after the triple end there is a polypeptide 18-pUC. It introduced the tetracycline resistance gene. from pBR322 in the corresponding direction (Reverse) of the CSF gene, 100 bases down from the 18-pUC polypeptide and the tetracycline resistance gene carries its own primer. Starting counterclockwise comes the gene for B lactamase, followed by pUC-18 (CoLEI), the origin of replication.

The final structural characteristic of the plasmid before returning to the GM-CST sequences is the primer PL. This initiator is essentially as described by A. skatzman and M. Rosenberg (in &Molecular cloning, a laboratory manual& (1982), Cold Spring

(419 Harbor Laboratory, page) and CSF transcription driven by the PL primer after heat induction in the appropriate E.Coli host strain.

The parental strain used for all strain combinations was w3110 lacl L8 (R. Brent and M. Ptashne PNAS 78 (1981) 4204-4208

It is completed by part λ. DNA nucleotides 34499 to 38214) chromosome W3110 lacl LS chromosome at the lacZ position. The integration occurred using the integration carrier consisting of pBR322 sequences carrying the genes for resistance to chloramphenicol and ampicillin in addition to the origin of replication (F. Bolivar Gene 4) (1978) 121-136) pBR322. The λ DNA fragment was inserted into the lacz gene, which itself is present. on the plasmid as a fragment extending from the BstFil locus in Laci to the TthiIII position downstream in lacz.

The integral of λ has been achieved. DNA was transferred to chromosomal copies of lacZ by homologous recombination. Lac colonies were found to be sensitive to ampicillin and resistant to chloramphenicol. A second recombination event was examined that removes all the excess plasmid sequences but leaves the λ portion. Integrated DNA - on lactose MacConkey strips. The first climatic type camR,ampS,lac changed to the climatic type -camS,amps,lac after the second recombination event. The resulting strain was named 400 GL, and its λR was at 30 and λS was at 42. Explains this

Climatic type: presence of functional chromosomal copies of the cl857 isotype.

GL400-PL was made by transformational induction of degradation products grown on strain (ul69, ara? 139rpsl lon? 100:: tnl0?SG20252 (lac).

Tn10 was processed by screening for Tets on selective media (S. Malloy, W. Nunn J. Bacteriol. 145 (1981).1110-1112)

It was called the final family line

, LacZ ? ⃰ (λCl, REX, N), lon? 100) (G1413) (JacloL

Converted Ptalc-185R to G1413. An overnight culture of this strain was grown at 30°C in 5 ml of induction medium containing 7 μg of 1-tetracycline. The induction medium contains per liter:

20 gm cosamino acids

06g Na2HPO47H2O

03 g kh2po4

0.5 g sodium chloride

Glycerol 1%

02 mg Vitamin B1

02 mg CaC12.2H2O

0.2 g MgCl2.6H2O

Dilute this medium (25 ml) containing 7 µg/ml tetracycline with 125 µl of overnight culture and shake at 30 C in a water bath until the culture reaches a density of 0.5A550 and quickly transfer (rapidly transfer) to a 40 C water bath and shake for an additional 2 hours. To allow GM-CSF synthesis. Cells were obtained and assayed to contain CSF by polyacrylamide gel electrophoresis for SDS. Under these conditions GM-CSF accumulates up to approximately 5% of the cellular protein.

example and

GM-CSF transcription in Saccharomyces cerevisiae

1- Installing the stand

The carrier containing the gene for the enzyme in the uracil biosynthesis pathway (URA3) was assembled as a test gene and 2 reproductive modules. This gene was derived from (1979) 24-17 Y1p5 (Botstein et al., Gene 8. pp) with the addition of a fragment containing the origin of replication from a 2-μm plasmid in yeast.

2- Isolation of the gene for glyceraldehyde phosphate dehydrogenase (GPDH)

Two GPDH genes were isolated from yeast (Holland and Holland Journal of Biological Chemistry 255 pp. 2596-2605 (1980). An oligonucleotide rod synthesized from the published sequence was used to isolate the GPDH gene from a plasmid of yeast nuclear DNA by typical methods. A plasmid containing the gene was deposited Previously complete GAP491 (ATCC No. 39777).

5- Preparation of glyceraldehyde phosphate dehydrogenase initiator

Phosphate Dehydrogenase (GPDH) for non-homologous gene transcription

A plasmid was constructed that allowed the natural insertion of the GPDH primer from the beginning of the desired non-homologous synthetic gene. This was done by inserting the position K pnl immediately close to the starting methionine codon in the synthetic GPDH gene. Then insert the ®ister&primer into the yeast Y opl transcription holder. 8- Isolation of the gene for factor alpha The gene for factor mating pheromone alpha was isolated from yeast (Kurjan and Herskowitz. Cell, Vol. 30, pp. 933-943 (1982))

A rod oligo nucleonide synthesized from this sequence was used to isolate the gene from plasmid DNA of the yeast nucleus by typical methods.

E- Preparation of GM-CSF cloning plasmid

From the elements described above, and the human CSF gene, the transcription carrier (AJ14, figure) was assembled in typical ways. Into this carrier the natural guide sequence for CSF was extracted and the sequence encoding the soon-to-be-mature CSF alpha-factor was inserted - only the sera were shortened. Between the GPDH primer and the pre-factor alpha sequence, the CSF exudate sequence was traced below it and confirmed by deoxynucleotide sequencing.

AAATAAACAAAATGCGTTTTCCTTCA.....AAA

AGA GAG GCG GAA GGT. GCA ccc GCC CGC TCG ...........

27- GM-CSF transcription

Transfer the plasmid AJ14 to a strain of Saccharomyces cerevisiae Cells and culture the cells to be CSF.

CSF cells were cultured to be