Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1995-05-23
2003-07-01
Yucel, Remy (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S350000, C530S351000, C435S069100, C435S069400
Reexamination Certificate
active
06586575
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
This invention discloses a commercially advantageous process for extraction and purification of protein from microorganisms. The initial steps of the process are useful for purifying many insoluble proteins while later steps are designed to renature denatured somatotropins produced by transformed microorganisms. The process is especially useful for purifying recombinantly-produced bovine somatotropin (rbSt).
Somatotropins are growth hormones which were originally discovered in pituitary gland extracts of various animals. In general, somatotropins are conserved molecules and similarities in amino acid sequence and structure are found between animals of disparate evolutionary ranking evidencing a common ancestral relationship.
Somatotropins are globular proteins comprised of a single chain of approximately 200 amino acids, having 2-3 intrachain disulfide bonds. Accordingly, bovine somatotropin (bSt) is comprised of a single chain of 190-191 amino acids, a globular structure with two intrachain disulfides, and a molecular weight of approximately 22,000 daltons.
BSt is able to remain insoluble in an aqueous environment when in the reduced state. This characteristic is exploited in the present invention as it relates to extraction and purification procedures. The disclosed process is applicable for other somatotropins having this same characteristic.
Growth hormones especially bSt have great commercial potential. Growth hormones in general have been shown to increase the growth rate of cattle, hogs and salmon, along with a significant increase in the meat to fat ratios. Moreover, bSt has value in its proven ability to increase milk production in dairy cattle. It is presently anticipated that 5-50 mg of growth hormone will be necessary per day per animal to achieve effective results.
Although a number of processes are presently available for the extraction of somatotropins from
E. coli
or other microorganisms on a laboratory scale, a process for the commercial production or large scale production of bioactive somatotropins, especially bST, was not available up until now. The disclosed process optimizes recovery of somatotropins from a microorganism when that microorganism has produced the somatotropin in predominantly precipitated form rather than as a soluble protein. These precipitated somatotropins are often deposited in refractile bodies within the microorganism. The refractile bodies are visible under a light microscope.
Somatotropins are not the only heterologous proteins to form refractile bodies inside microorganisms. Refractile bodies are often produced as a result of overproduction of a protein. They are thought to be an internal storage mechanism. Refractile bodies are commonly seen in microorganisms expressing heterologous proteins due to pathogenic conditions, mutations or recombinant genetic transformation.
Precipitated somatotropins are thought to be in a reduced state when found in refractile bodies of transformed microorganisms such as
E. coli.
Although expression of eukaryotic protein in
E. coli
or other prokaryote systems can yield a amino acid chain identical to the natural product often times the secondary and tertiary structure of the native protein cannot be forced or duplicated by the prokaryote. Unless the energy state of the native form forces the product to the native conformation, chemical and thermal intervention will be necessary to yield a biologically active and non-antigenic product.
BSt has 4 cysteine residues for which 3 different arrangements of disulfide bonds are possible in the oxidized monomeric state, only one of which is native. Upon workup without the precautions disclosed herein the cysteine residues will oxidize using available oxygen to form both polymeric and monomeric bSt with random disulfides. The random states so generated result in substantial yield reductions of bioactive growth hormones.
The classical studies of protein folding involving reduction and reoxidation of proteins were performed on enzymes such as ribonuclease (RNAse) and lysozyme. Givol, D., DeLorenzo, F., Goldberger, R. F., and Anfinsen, C. B., Biochemistry 53:676-684 (1965); and Saxena, V. P., and Wetlaufer, D. B., Biochemistry 9(25):5015-5022 (1970). The proteins are typically reduced with B-mercaptoethanol (BME) or dithiothreitol (DTT) in the presence of a denaturant such as 6-8 M urea or guanidine-HCl. The reductant and denaturant is then removed by gel filtration, and the protein is allowed to air-oxidize at low concentration.
The mechanism presently accepted for the reoxidation of reduced proteins involves rapid reoxidation of the protein to form random disulfide bonds, followed by disulfide interchange (“shuffling”). Although the protein may form incorrect disulfide bonds at first, these disulfides may be reduced again by reaction with free thiol groups on other proteins or thiol reagents. This gives each protein molecule the chance to assume a variety of disulfide bonding modes until the majority of the molecules are in the lowest energy configuration. The lowest energy state is usually the native conformation. Acharya, A. S., and Taniuchi, H., Molec. and Cell. Biochem. 44:129-148 (1982); and Anfinsen, C. B., Science 181:223-230 (1973).
Unlike the proteins described above, fully reduced bSt is insoluble in most aqueous solvents. Thus, many of the classical techniques for oxidation of reduced proteins are not applicable to bSt and the available methods taught in the literature concerning rbSt purification from
E. coli
were not practical for commercial applications.
The available literature discloses general principles of purification of heterologous proteins from microorganisms. Basically the steps involve cell kill, lysis, selective solubilizing of host debris, mechanical collection of the precipitated heterologous protein and solubilizing the heterologous protein followed by further filtration steps.
Prior to this invention, it was not possible to kill host cells without a concomitant precipitation of large amounts of undesired protein and other host cell debris. The precipitation of undesired protein along with desired insoluble proteins adds to the complexity of downstream purification procedures. This invention discloses the use of nonpolar organic solvents to kill cells. By eliminating standard killing steps using heat, phenol or combinations of phenol and toluene and by using the nonpolar organic solvents disclosed herein, it is possible to minimize unwanted host protein precipitation.
This invention also provides for an effective one step renaturation of somatotropins in the presence of detergents. Prior art recommendations suggest that renaturing of any desired protein to its native state had to be done at low concentrations of less than 1 mg/ml to avoid polymerization of bSt through cysteine residues. This invention resolves that problem by avoiding solubilization of somatotropins in nondetergent chaotropic agents such as urea or guanidine. By using mild detergents to solubilize the somatotropins, this invention demonstrates that it is possible to renature rbSt and like somatotropins at concentrations greater than 15 mg/ml.
The disclosed process has other advantages over the prior art processes. This process eliminates the need for thiol-reducing agents such as mercaptoethanol or glutathione. There is no need to use high concentrations of denaturants such as urea or guanidine to solubilize the desired proteins. Urea and guanidine have to be used at molar concentrations of up to 9 moles per liter and such concentrations will inevitably cause problems in large scale purifications resulting in increased production costs. This procedure also provides for alternatives to sodium dodecyl sulfate (SDS) as a solubilizing detergent which are advantageous because SDS is known to be difficult to remove from bSt. Dellacha, J. M., Annals N.Y. Acad. Sci., 148:313-327 (1968). One further advantage of this procedure is that bSt is split into two discrete populations upon removal of detergent. Upon removal of
Evans Timothy W.
Knuth Mark W.
Darnley , Jr. James D.
Pharmacia & Upjohn Company
Yucel Remy
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