Secretion of Mature IFN–α2 and Accumulation of Uncleaved Precursor by Bacillus subtilis Transformed with a Hybrid α–Amylase Signal Sequence–IFN–α2 Gene

Schein, Catherine H.; Kashiwagi, Kiyoko; Fujisawa, A.; Weissmann, Charles

doi:10.1038/nbt0886-719

Cited by 40 publications

(17 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Palva observed high levels of secreted a-amylase when the native gene from B. amyloliquefaciens was cloned into B. subtilis (19). By using these regulatory and secretory signals, respectable levels of the E. coli ,-lactamase (20 mg liter-' [21]) and human alpha-2 interferon (500 ,ug liter-' [20,23]) were also obtained. Other secretion signals that have been studied include those of the penP gene from B. licheniformis (3) and the ax-amylase (17,25) and protease (30) genes from B. subtilis.…”

mentioning

confidence: 94%

Secretion and processing of staphylococcal nuclease by Bacillus subtilis

Miller¹,

Kovacevic²,

Veal³

1987

J Bacteriol

View full text Add to dashboard Cite

We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis. We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B. This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease. The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments. The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A descriled for S. aureus Foggi. The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide. However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease.

show abstract

mentioning

confidence: 94%

Secretion and processing of staphylococcal nuclease by Bacillus subtilis

Miller¹,

Kovacevic²,

Veal³

1987

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Because much of this interest is focused on proteins which are normally secreted from animal cells, development of such bacterial strains is based on the premise that a protein which is normally secreted from an animal cell can also be exported from a bacterial cell. Although there have been a few reports of export of animal proteins into the medium by bacteria in culture, these proteins have generally been small, e.g., interferon (23,30,33) and human growth hormone (11) or have been peptides fused to carrier bacterial proteins (21,41).…”

mentioning

confidence: 99%

Secretion of human serum albumin from Bacillus subtilis

Saunders¹,

Schmidt²,

Mallonee³

et al. 1987

J Bacteriol

View full text Add to dashboard Cite

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.

show abstract

“…However, some proteins seem to be efficiently secreted while others are poorly secreted. When human interferon a-2b was expressed in B. subtilis and secreted with the help of the a-amylase signal sequence, large amounts of the uncleaved precursor protein were accumulated in the cells (28). B. subtilis secretes several proteases into the culture medium which can potentially degrade the recombinant proteins that are also secreted into the medium.…”

Section: Bacillus Subtilismentioning

confidence: 99%