Secretion of Interferon by Bacillus subtilis

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.

mentioning

confidence: 99%

Secretion of human serum albumin from Bacillus subtilis

Saunders¹,

Schmidt²,

Mallonee³

et al. 1987

“…A large number of both procaryotic and eucaryotic genes have been successfully fused to the regulatory regions and signal peptide sequences derived from the Bacillus system for expression and secretion studies (3,4,6,9,10,12). The reported levels of secreted proteins ranged from less than 1 to 50 mg/liter for rat proinsulin and staphylococcal nuclease, respectively.…”

mentioning

confidence: 99%

“…The reported levels of secreted proteins ranged from less than 1 to 50 mg/liter for rat proinsulin and staphylococcal nuclease, respectively. In most of the reported cases, the low yield and poor quality of secreted heterologous proteins were mainly due to the simultaneous production of at least three extracellular proteases (neutral protease, subtilisin, and esterase) during the sporulation or stationary phase (1,3,9,15). These proteases significantly reduced both the physical integrity and the biological activities of the secreted foreign proteins.…”

mentioning

confidence: 99%

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth

Wong¹,

Kawamura²,

Doi³

1986

We report the development of an efficient Bacilus subtiis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain.Bacillus subtilis serves as an excellent system for studying the expression and secretion of foreign proteins, since B. subtilis is able to secrete a wide variety of enzymes, such as proteases, a-amylase, and 3-glucanase, directly into the medium at high levels (8,13,17,18). A large number of both procaryotic and eucaryotic genes have been successfully fused to the regulatory regions and signal peptide sequences derived from the Bacillus system for expression and secretion studies (3,4,6,9,10,12). The reported levels of secreted proteins ranged from less than 1 to 50 mg/liter for rat proinsulin and staphylococcal nuclease, respectively. In most of the reported cases, the low yield and poor quality of secreted heterologous proteins were mainly due to the simultaneous production of at least three extracellular proteases (neutral protease, subtilisin, and esterase) during the sporulation or stationary phase (1, 3, 9, 15). These proteases significantly reduced both the physical integrity and the biological activities of the secreted foreign proteins. In this report we describe the use of a combination of a secretion vector system, an improved double protease mutant strain (5), and an enriched medium to achieve efficient synthesis of biologically active TEM P-lactamase, which can be stably maintained in the growth medium for at least 100 h without any significant degradation.With the in vitro and in vivo characterization of both the transcription regulatory region (16) and the signal peptidase cleavage site for the subtilisin gene (1Sa), we were able to dissect the transcription regulatory element and the signal peptide sequence from the subtilisin gene and fuse them to a DNA sequence encoding the mature ,B-lactamase enzyme in a pUB110 derivative plasmid to form the pSHL-7 vector. The detailed construction of pSHL-7 plasmid has been reported elsewhere (1Sa). This vector was used to study the effect of host proteases on the stability of secreted ,3-lactamase. The pSHL-7 plasmid was transformed into isogenic B. subtilis DB1l1 (his), DB102 (his nprR2 nprE18), and DB104 (his nprR2 nprE18 aprA3). The cells were grown in the 2XSG sporulation medium (7), and the secreted ,B-lactamase activity was determined following growth by using PADAC (CalBiochem) as the P-lactamase substrate.As shown in Fig. 1 and Table 1, the peak level of secreted ,B-lactamase activity from strain DB102 was slightly higher than that from strain DB11. However, both showed much * Corresponding author. lower activity than DB104. At 5 h after the cell enters the stationary phase, the P-lactamase activity from DB1l1 and DB102 dropped significantly, almost to the background level, w...

“…In fact, this observation is reminiscent of chicken ovalbumin (11) and rat preproinsulin (34,35) export in E. coli and interferon export in Bacillus subtilis (27). It was surprising, however, to find that the P-galactosidase signal peptide was sufficient to direct the export of E. coli galactokinase.…”

Section: Discussionmentioning

confidence: 97%

Secretion of interleukin-1 beta and Escherichia coli galactokinase by Streptomyces lividans

Lichenstein¹,

Brawner²,

Miles³

et al. 1988

The functionality of the Streptomyces lividans beta-galactosidase signal peptide to direct heterologous protein export was examined. The signal peptide plus eight amino acids of mature protein were sufficient to export not only a naturally exported protein, interleukin-1 beta, but also a naturally occurring cytoplasmic protein, Escherichia coli galactokinase. Interestingly, cells which expressed yet exported galactokinase were phenotypically Gal-. The potential use of the exported galactokinase system for the isolation and characterization of mutations within signal peptides and the export machinery of the host is discussed.