Use of Bacteriocin Release Protein in E. Coli for Excretion of Human Growth Hormone into the Culture Medium

Because of the outer membrane barrier, the gram−negative bacterium E. coli is generally not considered to be an appropriate host for the excretion of proteins into the culture medium. However, we have been able to efficiently excrete a mammalian protein, human growth hormone (hGH), into the E. coli culture medium using E. coli cells harboring two vectors, the hGH secretion vector pOmpA−hGH2 and the pCloDF 13−derived bacteriocin release protein (BRP) expression vector pJL3. Both vectors contain, in tandem, the E. coli lpp and the E. coli lac promoter−operator system. The tandem promoter drives the expression of BRP and hGH and the expression of both proteins is regulated by the E. coli lac represser. In the presence of isopropyl−1−thio−−D−galactopy ranoside (IPTG) at a low concentration (20 M), both the BRP and the OmpA−hGH hybrid protein were expressed and mature hGH was released into the culture medium. The excreted hGH contained the correct amino terminus corresponding to the amino terminus of hGH derived from the human pituitary gland, indicating the correct processing of the OmpA signal peptide. The excretion of hGH was efficient and it was purified from the culture medium to greater than 98% purity using a single step of reversed−phase column chromatography.

1. Hsiung, H.M., Mayne, N.G., and Becker, G.W. 1986. High-level expression, efficient secretion and folding of human growth hormone in Escherichia coli. Bio/technology 4:991–995. | ISI | ChemPort |
2. Becker, G.W. and Hsiung, H.M. 1986. Expression, secretion and folding of human growth hormone in Escherichia coli. Purification and characterization. FEES Letters 204:145–150. | Article | ChemPort |
3. Chang, C.N., Rey, M., Bochner, B., Heyneker, H., and Gray, G. 1987. High-level secretion of human growth hormone by Escherichia coli. Gene 55:189–196. | Article | PubMed  | ISI | ChemPort |
4. Mackman, N., Baker, K., Gray, L., Haigh, R., Nicaud, J.M., and Holland, I.B. 1987. Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of hemolysin. EMBO J. 6:2835–2841. | PubMed  | ISI | ChemPort |
5. Abrahmsén, L., Moks, T., Nilsson, B., and Uhlén, M. 1986. Secretion of heterologous gene products to the culture medium of Escherichia coli. Nucleic Acids Res. 14:7487–7500. | PubMed  | ISI | ChemPort |
6. Nagahari, K., Kanaya, S., Munakata, K., Aoyagi, Y., and Mizushima, S. 1986. Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human -endorphin. EMBO J. 4:3589–3592. | ISI |
7. Kato, C., Kobayashi, T., Kudo, T., Furusato, T., Murakami, Y., Tanaka, T., Babe, H., Oishi, T., Ohtsuka, E., Ikehara, M., Yanagida, T., Kato, H., Moriyama, S., and Horikoshi, K. 1987. Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli. Gene 54:197–202. | PubMed  | ISI | ChemPort |
8. Kobayashi, T., Kato, C., Kudo, T., and Horikoshi, K. 1986. Excretion of the penicillinase of an alkalophilic bacillus sp. through the Escherichia coli outer membrane is caused by the insertional activation of the kil gene in plasmid pMB9. J. Bacteriology 166:728–732. | ISI | ChemPort |
9. de Graaf, F.K. and Oudega, B. 1986. Production and release of cloacin DF13 and related colicins. Current Topics in Microbiol. and Immunol. 125:183–205. | ChemPort |
10. Luirink, J., de Graaf, F.K., and Oudega, B. 1987. Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli. Mol. Gen. Genet. 206:126–132. | PubMed  | ISI | ChemPort |
11. Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Masui, T., and Inouye, M. 1984. Secretion cloning vectors in Escherichia coli. EMBO J. 3:2437–2442. | PubMed  | ISI | ChemPort |
12. Uhlin, B.E., Molin, S., Gustafsson, P., and Nordstrom, K. 1979. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene 6:91–106. | Article | PubMed  | ISI | ChemPort |
13. Chang, A.C.Y. and Cohen, S.N. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141–1156. | PubMed  | ISI | ChemPort |
14. Oudega, B., Stegehuis, F., and de Graaf, F.K. 1983. Effect of glucose fermentation on the functioning of protein H in the excretion of cloacin DF13 by Escherichia coli. FEMS Microbiol. Letters 21:125–131. | Article | ISI |
15. Bewley, T.A. and Li, C.H. 1975. The chemistry of human pituitary growth hormone. Adv. in Enzymol. 42:73–166. | ISI | ChemPort |
16. Schoner, R.G., Ellis, L.F., and Schoner, B.E. 1985. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154. | ISI | ChemPort |
17. Maurer, R., Meyer, B.J., and Ptashne, M. 1980. Gene regulation at the right operator (Or) of bacteriophage J. Mol. Biol. 139:147–161. | PubMed  | ISI | ChemPort |
18. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitro cellulose. Gene Anal. Techn. 1:3–8. | Article | ChemPort |
19. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 277:680–685. | ISI |
20. Somack, R. 1980. Complete phenylthiohydantoin amino acid analysis by high-performance liquid chromatography on ultrasphere-octade-cylcrimethyloxysilane. Analytical Biochemistry 104:464–468. | PubMed  | ISI | ChemPort |
21. Pierce Chemical Company, 1985–86. Handbook and General Catalog.

	Top

	Top