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

Abstract

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–β–D–galactopy ranoside (IPTG) at a low concentration (20 μM), 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.