Insulin-like growth factor-1 belongs to a heterogeneous family of peptides which share some of the biological and chemical properties of insulin, but which are antigenically distinct from insulin. Currently available experimental evidence suggests that IGF-1 promotes growth by mediating the effects of growth hormone. Thus, such processes as skeletal growth, call replication and other growth related processes are affected by IGF-1 levels. Physiological concentrations of IGF-1 have been shown to be influenced by such conditions as thyroid disease, diabetes and malnutrition (see Preece (1983) Horm. Blood, 4: 108). IGF-1 has also been shown to act synergistically with other growth factors, for example, in accelerating the healing of soft and mesenchymal tissue wounds (see Lynch et al. (1989) J. Clin., Periodontol, 16: 545; and Lynch et al. (1987) Proc. Nat. Acad. Sci. USA, 84: 7696), and in enhancing the growth of mammalian cells in serum-free tissue culture medium (see Burleigh et al. (1986) American Biotech. Lab., 4: 48).
Considering the many clinical and research applications of IGF-1, a ready supply of IGF-1 would 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 IGF-1.
Among host cells that have been used for the production of heterologous proteins, E. coli and Saccharomyces cerevisiae (Baker's yeast) are probably the best characterized. Insulin-like growth factor-1 (IGF-1), which is a polypeptide of 70 amino acids with a molecular weight of 7648 daltons, is a single chain protein that has three intrachain disulfide bridges. These disulfide bonds, along with numerous hydrogen bonds and hydrophilic interactions, maintain the compact tertiary structure of this molecule. E. coli, however, does not possess the ability to produce disulfide bonds in proteins, so that proteins, such as IGF-1, that include disulfide bonds, when cloned into and expressed in E. coli, frequently are not stable and tend to aggregate into inactive complexes. In addition, IGF-1 produced in E. coli has to be extracted and treated with oxidizing agents to produce the disulfide bonds. Upon reduction and reoxidation, IGF-1 refolds in a variety of ways, forming as many as 15 monomeric configurations (Meng et al. (1988) J. Chrom., 443: 183) because cell breakage and too rapid formation of disulfide linkages results in random disulfide bond formation. In order to produce biologically active IGF-1, the resulting mixture of 15 different forms of IGF-1 must be separated. Consequently, the yield of purified product is very low (Grossgian (1985) Gene, 18: 199).
Furthermore, since E. coli is a prokaryote, in order to produce IGF-1 molecules which contain the authentic N-terminal glycine, and not the initiating methionine present on the primary translation product, it is necessary to express IGF-1 in E. coli as a fusion protein. Cleavage of mature IGF-1 from the initially produced fusion protein necessitates an additional step in the production process. Consequently, attempts to produce this peptide by recombinant means in E. coli host expression systems results in a complex mixture of product forms which must be separated for further use (see, Grossgian (1985) Gene, 18: 199).
Eukaryotic host cells, including yeast cells, thus, are the host cells of choice for the expression of many eukaryotic proteins. Yeast host cells offer clear advantages over bacteria in the production of heterologous proteins, including their ability to properly process pre-pro-heterologous proteins and secrete heterologous proteins into the culture medium. Secretion of proteins from cells is often superior to production of proteins in the cytoplasm because 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 addition, the secretory pathway of the cell and the extracellular medium tend to be oxidizing environments which support disulfide bond formation necessary for proper folding of many proteins (Smith, et al, (1985) Science 229: 1219); whereas, the cytoplasm is a reducing environment in which disulfide bonds do not form. Thus, for production of sulfhydryl-rich proteins that rely on disulfide bonds to maintain the correct tertiary structures, there is a compelling need to develop eukaryotic hosts capable of secreting such proteins into the culture medium. Therefore, production of sulfhydryl-rich proteins, such as IGF-1, that contain appropriately formed disulfide bonds, can best be achieved by transit through the secretory pathway.
IGF-1 has been cloned into and expressed using S. cerevisiae host cells by introducing DNA encoding IGF-1 on autonomously replicating extrachromosomal elements. Gellerfors et al. ((1989) J. Biol, Chem., 264: 11444-11449) describes the production of IGF-1 in S. cerevisiae under the control of the S. cerevisiae actin promoter. The IGF-1 product is encoded by autonomously replicating plasmid-borne DNA. In a similar study, Bayne et al. ((1988) Gene 66: 235-244) describes the production of IGF-1 in S. cerevisiae under the control of the S. cerevisiae alpha mating factor promoter. The latter, however, reports yields of IGF-1 of only about 2 mg of IGF-1 per liter of fermentation broth.
In view of this low yield and the problems generally encountered with up-scaling the production of heterologous proteins in autonomous plasmid-based yeast systems, such as loss of selection for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors operated at high cell density, there is a need to develop more efficient means for producing large quantities of biologically active IGF-1.
Therefore, it is an object of this invention to provide host cells and expression vectors that stably express IGF-1 and that secrete high concentrations of biologically active IGF-1.
It is another object of this invention to provide an expression system for the production of biologically active IGF-1 that, not only secretes high concentrations of biologically active IGF-1, but that can be readily scaled up to produce large quantities of such IGF-1.