Secretion of human serum albumin from Bacillus subtilis

Abstract

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.

Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae

“…This indicates that the protein is probably secreted from a periplasmic pool. A similar phenomenon has been reported in the case of secretory expression of proteins in Bacillus subtilis and also in the case of expression of LTB in Vibrio cholerae [6,17,18]. Proteins that are secreted with the help of the signal sequences follow the type II pathway of secretion and involve a periplasmic intermediate [19].…”

The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis

“…HSA (pI 5.2) exhibited in itro wall-binding characteristics that were comparable with those of AmyL, suggesting that, on the basis of electrostatic interactions at least, the wall is unlikely to represent a significant barrier to the secretion of HSA [18]. Instead, cell-associated degradation could account for the failure to detect significant amounts of this protein in the culture medium [18], as was the case for some of the chimaeras of AmyL [5].…”

“…Instead, cell-associated degradation could account for the failure to detect significant amounts of this protein in the culture medium [18], as was the case for some of the chimaeras of AmyL [5].…”

“…The assay was used to determine the wall-binding characteristics of wild-type AmyL and the charge-modified chimaeras. In addition, the wall binding of HSA (pI 5.2 ; see [38]), an eukaryotic protein whose secretion from B. subtilis has been suggested to be blocked by interaction with the cell wall [18], was also tested.…”

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes

“…Efficient expression of foreign genes can be obtained in B. subtilis by fusing the gene of interest inframe to regulation signals of an endogenous B. subtilis gene. Secretion vectors which use the regulatory signals of B. subtilis a-amylase (Sarvas, 1986 ;Schein et al, 1986 ;Saunders et al, 1987) and subtilisin (Vasantha & Thompson, 1986;Saunders et al, 1987;Wang et al, 1988) have been constructed, as well as an inducible system using the B . subtilis levansucrase gene promoter and the region encoding its signal sequence (Joliff et al, H .…”