Aprotinin is a polypeptide of 58 amino acids with a molecular weight of 6512 daltons. This single chain protein has three disulfide bridges, linking cysteines at position (5-55), (14-38), and (30-51). These disulfide bonds, along with numerous hydrogen bonds and hydrophilic interactions, maintain the compact acid-stable tertiary structure of this molecule. Aprotinin has been well studied because of its small size, tremendous stability and function.
Aprotinin is a potent, naturally occurring enzyme inhibitor which inhibits proteolytic enzymes of the serine protease family. It is commonly isolated from a variety of bovine tissues including lung, parotid gland, spleen, liver, and pancreas. Specific enzymes known to be inhibited by aprotinin include trypsin, chymotrypsin, kallikrein, and plasmin. Aprotinin is also known as Bovine Pancreatic Trypsin Inhibitor (BPTI) and Trypsin Kallikrein Inhibitor (TKI).
Aprotinin is the active ingredient of a drug which has found wide use for the treatment of traumatic hemorrhagic shock, inhibition of plasmin, preservation of blood cell function in acid citrate dextrose (ACD)-stored blood as well as many other applications. In general, aprotinin is useful in reducing pathologically elevated proteolytic enzyme activity to normal levels.
Aprotinin is also useful in preventing proteolytic damage of recombinant products, such as proteins, polypeptides, enzymes, proenzymes, and preprotein, during purification and isolation procedures. The optimum amount of aprotinin for use in total inhibition of proteinases must be determined on a case-by-case basis; however, micromolar concentrations of aprotinin are usually sufficient.
Aprotinin is used extensively in radioimmunoassays to prevent proteolytic damage of the ligand. Some of the radioimmunoassays that use aprotinin as a proteolytic enzyme inhibitor are assays of adrenocorticotropin, calcitonin, beta endorphin, glucagon, and renin somatostatin.
Considering the many clinical and research applications of aprotinin, a ready supply of aprotinin, such as that which would result from fermentation of aprotinin-expressing recombinant organisms, will be of great value to the medical and biotechnology fields.
Since isolation from natural sources is technically difficult, expensive, and time consuming, recent efforts have centered on the development of efficient recombinant methods for the production of APR.
Of the hosts widely used for the production of heterologous proteins, probably E. coli and Saccharomyces cerevisiae (Baker's yeast) are the best understood. However, E. coli tends to produce disulfide-bonded proteins such as APR in their reduced forms which frequently are not stable in the presence of endogenous bacterial proteases, and which tend to aggregate into inactive complexes. Attempts to overcome this problem, e.g., by employing a suitable leader sequence in order to produce soluble APR which could be readily recovered from the cell broth, resulted in other inconveniences, especially during purification of the product, since the bulk of the desired protein was associated with the cell paste.
For example, Auerswald, Schroder and Kottich, in Biol. Chem. Hoppe Seyler (Germany, West) 368, 1413-1425 (1987) disclose the synthesis, cloning and expression of recombinant aprotinin in E. coli using a synthetic gene formed by the fusion of DNA fragments of the aprotinin coding sequence. Product was expressed as a fusion protein with beta-galactosidase. Cyanogen bromide cleavage of the fusion protein, followed by purification and renaturation is required to produce biologically active product.
Marks, et al., in J. Biol. Chem. 261, pp. 7115-118 (1986), report the successful expression of bovine pancreatic trypsin inhibitor (BPTI) in E. coli. These workers were able to overcome the difficulties in producing disulfide-bonded proteins in E. coli, describing the production of correctly folded BPTI that was conformationally indistinguishable from native BPTI.
Other publications dealing with the recombinant production of aprotinin include European Patent Application Nos. 238,993, 244,627, and 208,539.
Yeasts can offer clear advantages over bacteria in the production of heterologous proteins, which include their ability to secrete heterologous proteins into the culture medium. Secretion of proteins from cells is generally superior to production of proteins in the cytoplasm. Secreted products are obtained in a higher degree of initial purity; and further purification of the secreted products is made easier by the absence of cellular debris. In the case of sulfhydryl-rich proteins, there is another compelling reason for the development of eukaryotic hosts capable of secreting such proteins into the culture medium: their correct tertiary structure is produced and maintained via disulfide bonds. This is because the secretory pathway of the cell and the extracellular medium are oxidizing environments which can support disulfide bond formation [Smith, et al., Science, 229, 1219 (1985)]; whereas, in contrast, the cytoplasm is a reducing environment in which disulfide bonds cannot form. Upon cell breakage, too rapid formation of disulfide linkages can result in random disulfide bond formation. Consequently, production of sulfhydryl-rich proteins, such as APR, containing appropriately formed disulfide bonds, can be best achieved by transit through the secretory pathway.
Secretion of aprotinin having the desired intramolecular disulfide bonds from E. coli is suggested in Japanese document No. 63230089 (assigned to Takeda Chemical Ind KK). The cited document contains no details as to the level of secretion or the purity of APR obtained, nor is there any suggestion that APR could be expressed in any other host system besides E. coli.
PCT International Application No. PCT/DK88/00138, bearing International Publication No. WO 89/01968, describes the production of aprotinin and aprotinin homologues in S. cerevisiae under the control of the S. cerevisiae triosephosphate isomerase promoter. The aprotinin and aprotinin homologues are encoded by autonomously replicating plasmid-borne DNA. The reported yields of aprotinin and homologues thereof are quite low, ranging from 1-13 mg of aprotinin per liter of fermentation broth.
In view of the problems usually encountered with up-scaling the production of heterologous proteins in autonomous plasmid-based yeast systems, such as S. cerevisiae, and the low expression levels of aprotinin and aprotinin homologues achieved in reported work in S. cerevisiae, no motivation is provided by the art for one to further pursue the production of APR in S. cerevisiae. To overcome the major problems associated with the expression of recombinant gene products in S. cerevisiae (e.g., loss of selection for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors operated at high cell density), a yeast expression system based on methylotrophic yeast, such as for example, Pichia pastoris, has been developed. A key feature of this unique system lies with the promoter employed to drive heterologous gene expression. This promoter, which is derived from a methanol-responsive gene of a methylotrophic yeast, is frequently highly expressed and tightly regulated (see, e.g., European Patent Application No. 85113737.2, published Jun. 4, 1986, under No. 0 183 071 and issued in the U.S. on Aug. 8, 1989, as U.S. Pat. No. 4,855,231). Another key feature of expression systems based on methylotrophic yeast is the ability of expression cassettes to stably integrate into the genome of the methylotrophic yeast host, thus significantly decreasing the chance of vector loss.
Although the methylotrophic yeast P. pastoris has been used successfully for the production of various [Cregg et al., Bio/Technology 5, 479 (1987)], lysozyme and invertase [Digan et al., Developments in Industrial Microbiology 29, 59 (1988); Tschopp et al., Bio/Technology 5, 1305 (1987)], endeavors to produce other heterologous gene products in Pichia, especially by secretion, have given mixed results. At the present level of understanding of methylotrophic yeast expression systems, it is unpredictable whether a given gene can be expressed to an appreciable level in such yeast or whether the yeast host will tolerate the presence of the recombinant gene product in its cells. In addition, it is unpredictable whether desired or undesired proteolysis of the primary product will occur, and if the resulting proteolytic products are biologically active. Further, it is especially difficult to foresee if a particular protein will be secreted by the methylotrophic yeast host, and if it is, at what efficiency. Even for the non-methylotrophic yeast S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.