Method of lipophilic proteins extraction

This invention relates to the production of a protein and its purification. In one aspect, the invention relates to the separation of soluble protein from broken cells. In another aspect, the invention relates to selective extraction of proteins.

Recombinant DNA technology is rapidly becoming an effective tool for the production of pepids and proteins that are of interest in various diagnostic, therapeutic and chemical applications, etc. One problem with which technology is often encountered is the need to obtain the desired protein in purified form without the deleterious proteins which also occur during the expression of the desired product. The present invention is directed to improved methods for obtaining proteins produced by recombinant DNA technology. It is therefore an object of the present invention to provide a method of effectively recovering the desired proteins produced from genetically modified yeast organisms.

This and other objects of the invention will become apparent from the following description and appended definitions of the invention. 3

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In accordance with the invention, lipophilic proteins produced by genetically modified strains of Pichla can be selectively obtained using a lye paste containing chaotropic salts. When digesting the strains of the strains in the presence of such lysing buffers, the total amount of the extracted protein is reduced, with little effect on the extraction of the desired lipophilic proteins. Soluble cell extracted in the present invention thus contains an increased concentration of lipophilic protein relative to all-other proteins contained in the extract, thereby simplifying further purification steps to subject the desired lipophilic protein.

The soluble fraction isolated by the process of the present invention can be further processed by techniques known in the art to further concentrate and purify the desired protein. Thus, soluble protein can be concentrated by dialysis, passage through the cross-linking and reverse phase and subsequent elution with a minimum volume of solvent, precipitation, ultrafiltration, lyophilization, and the like. size exclusion, high performance liquid chromatography (HPLC), ion exchange, hydrophobic chromatography, and the like.

As used herein, the term &amp; quot; lipophilic protein &amp; quot; is used for proteins that tend to associate with lipid membranes or which are in the presence of lipid-like or lipid-like structures in micelle-like structures.

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O - 5 - proteins usually contain a high proportion of hydrophobic amino acids, isoleucine, valine, leucine, phenylalanine, tryptophan and alanine.

Examples of lipophilic proteins to which this term is not limited are all forms of hepatitis B surface antigen, including S-form, pre-form, pre-form, and the like, phosphatidylserine decarboxylase (from E. coli) a lambda hakteriophage D-protein; low density lipoprotein (LDL); high density lipoprotein (HDL) adihydrocoronate dehydrogenase.

The term &amp; quot; chaotropic salt &amp; quot ;, as used herein, refers to salts whose anions promote the transfer of apolar groups into water, which include compounds containing thiocyanate, halide ions such as iodide and bromide, halo-ionic ions such as hypochlorite ions, lithium, calcium and water - 6 -

Examples of chaotropic salts useful in the process of the invention are sodium thiocyanate, potassium thiocyanate, sodium iodide, potassium iodide, sodium hypochlorite, chloride lithium, lithium bromide, guanidinium hydrochloride, guanidinium thiocyanate, urea, and the like.

The production of lipophilic proteins by Piice can be carried out by genetic modification of the appropriate Pichia host strains using the DNA sequences encoding the desired protein. Suitable DNA sequences encoding the desired lipophilic protein are readily available to those skilled in the art, for example by isolation of natural sources, by the construction of a synthetic DNA sequence and the like. Specific DNA manipulation techniques in Pichia strains are disclosed in articles in Volume 5 of Molecular and Cellular Biology, pp. 1111 and 3376 (1985)

The method of extraction according to the invention is carried out by subjecting the cells to conditions causing their breakdown for a time sufficient to break substantially all the cells, the breakage being carried out in the presence of an extraction medium containing at least one chaotropic salt in about one-fold to about 8-millimolar concentration in a pH-7 medium which is suitable for maintaining the desired protein in a stable form, usually at a pH in the range of about 6 to about 8. In order to minimize the degradation of the protein during the extraction process, antiprotease agents may optionally be added to the buffer, such as phenylmethylsulfonyl fluoride.

In order to break the cells, homogenization in a ball mill or similar device is generally employed in the practice of the present invention. The time required for crushing the cells is dependent on the sensitivity of the cell walls to breakage, sharpness of homogenization conditions, the presence and concentration of the components in the lyside phytrue, etc. Usually, the cells undergo conditions causing their breakdown for a time ranging from about 0.5 to about 30 minutes, for a time ranging from about 1 to about 5 minutes.

The temperature used to break the buckets is usually regulated to minimize the action of enzymes degrading enzymes. The breakage of the cells is usually carried out at a temperature in the range of about 0 to about 10 ° C, preferably about 0 ° C. This minimizes the extent of degradation of the desired protein during the cell grafting step and increases the yield of the desired protein isolated from the crushed cells.

After crushing the cells, the dissolution / perfusion is isolated by separating the broken cells by techniques known in the art, for example by centrifugation by non-rotational filtration. The resulting cell-free liquid is richer with the desired lipophilic protein as compared to the liquid obtained by simply crushing the cells and isolating the soluble fraction.

If desired, the resulting liquid may be subjected to further processing in order to obtain a concentrated proportion of the desired lipophilic protein by various techniques known in the art such as acid precipitation, filtration, chromatography, solvent evaporation, and the like.

The invention is illustrated by the following examples. The examples are illustrative only and the scope of the invention is not to be construed in any way. - 9 -

Example I

Hepatitis B surface antigen (HBsAg) extraction in the form of 22nm particles of phyia cells transformed with the pBSAGI5I vector (available in the E. coli host at the US Department of Agriculture Research Institute, Seoria Regional Research Center of the US Department of Agriculture, Peoria, Illinois) under the registration number NNRL B-18021 is performed as follows:

P. Pastoris cultures do not grow to a cell density in the range of 10 to 100 units of optical density (600 nm) per millimeter. An aliquot of 100 units of optical density is transferred to a 13 x 100 mm bidosilicate culture tube twice with 20 volumes of lysine buffer (the composition shown below). In the pelletised cells (using the IEC clinical centrifuge), 0.5 g of acid-laced glass beads of 0.5 mm diameter and then 0.35 ml of lysine buffer are added. The lysine buffer contains either 0.5 M NaCl and 0.1 Triton X-100 (w / v) in the control buffer or 2M or 3M concentration of cha-10

tropic salts in the presence or absence of 0.1% Triton X-100. All solutions were quenched to pH 7.5 with 10 mM sodium phosphate. The mixture is stirred for one minute at a maximum speed using a vortex mixer. Between mixing stirrers, the mixture is cooled on ice for at least one minute. During vortexing, the tubes are preferably maintained at a temperature of 20-40 ° to achieve maximum cell breakdown performance. Upon completion of the lysis the solution of the crushed cells is separated, the glass beads washed with 0.35 ml of lysine buffer and the two solutions are combined and centrifuged for 15 minutes at 13000 x g Supernatants are separated and tested for immunoreactive HBsAg particles (Ausria assay) and total protein (Bradford). The results are shown in Table I (a).

Example II

To further illustrate the method of the present invention, 80 ml of suspension (one volume of agglutinated cells per two volumes of lysine buffer) is subjected to a cell disintegration device (Impandex lne.) Using a 64 mm stirring disk at a freq- 4500 min. The lysine buffer contains either 11 - 0.5M NaCl and 0.1 Triton (w / v) or 3MKSCN. Supernatants were tested for HBsAg (Ausria) and total protein content (Bradford). The results are shown in Table I (b).

Table I

I II III

Conditions for the HBsAg lysate all HBsAg / protein (yug / ml) protein (mg / ml) (w / w). (a) salt (end). NaCl (0.5M) + Triton 230 10.1 2.3 KI (2M) + Triton 14 1.4 1.0 KI (2M) - Triton 169 2.4 7.0 KSCN (3M) + Triton &lt;10 1 , 9 &amp; lt; 0.5 KSCN (3M) - Triton 222 3.5 6.3 (b) Whipping Equipment

NaCl (0.5M) + Triton 600 32.5!, 8 KSCN (3M) 803 10.6 7.6-12

While none of the conditions in the case of the use of the chaotropic salt (KI or KSCN) results in significantly higher HBsAg particles compared to the control experiment (Column I), it is clear that chaotic tropic salts inhibit the release of all protein (Column II) increase the HBsAg specific activity of two to five times (column III). in