Bacillus protein secretion: an unfolding story (original) (raw)
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PROTEOMICS, 2009
Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin-arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper-producing strain. While LipA is secreted Secdependently under standard conditions, hyper-produced LipA is secreted predominantly Tat-dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper-produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec-dependently transported proteins have potential twin-arginine signal peptides for export via the Tat pathway.
Essays in Biochemistry, 2021
Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium’s intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into th...
Recombinant protein secretion in Escherichia coli
Biotechnology Advances, 2005
The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.
SecA protein is directly involved in protein secretion in Escherichia coli
FEBS Letters, 1989
A high‐expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.
Post-translocational folding of secretory proteins in Gram-positive bacteria
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2004
The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Grampositive bacteria such as Bacillus subtilis encode membrane-and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.
Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis
Research in Microbiology, 2004
Bacillus subtilis is an alternative host for expression and secretion of heterologous proteins. However, low yields of protein production limit its use on a wide scale. The secretory pathway of proteins can be divided into three functional stages: the early stage, involving the synthesis of secretory pre-proteins, their interaction with chaperones and binding to the secretory translocase; the second stage, translocation across the cytoplasmic membrane; and the last stage, including removal of the signal peptide, protein refolding and passage through the cell wall. Five bottlenecks for expression and secretion of heterologous proteins are described in this review: transcription, protein folding, translocation, signal peptide processing and proteolysis. 2004 Elsevier SAS. All rights reserved.
Engineering Signal Peptide for Enhanced Protein Secretion in Lactococcus
Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and 15 clinical delivery of therapeutics. In many such applications using this host, it is desirable to 16 maximize secretion of recombinant proteins into the extracellular space, which is typically 17 achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In 18 order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal 19 peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to 20 probe the effects of both mRNA sequence (silent mutations) and peptide sequence (amino acid 21 substitutions) on secretion. We screened signal peptides based on their resulting secretion levels 22 of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis 23 α-amylase. Silent mutations alone gave up to a 16% increase in the secretion of α-amylase 24 through a mechanism consistent with relaxed mRNA folding around the ribosome binding site 25 and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with 26 additional silent mutations from the best clone in the initial screen, yielded up to a 51% increase 27 in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The 28 best sequence from our screen preserves the tripartite structure of the native signal peptide but 29 increases the positive charge of the n-region. Our study presents the first example of an 30 engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more 31 generally, provides strategies for further enhancing protein secretion in bacterial hosts. 32 vehicle because it can survive passage through the stomach acid and contact with bile (8, 9). L. 48 lactis has been engineered to express and secrete a variety of therapeutic proteins, including 49 interleukin-10 for treatment of inflammatory bowel disease (10), bovine beta-lactoglobulin for 50 immunity against this cow milk's allergen (11), and single-chain insulin for treatment of diabetes 51 (12). In such biomedical applications, it is often desirable to maximize secretion of the 52 recombinant protein into the extracellular space in order to reduce the dose and administration 53 frequency of the therapeutic bacteria. 54 55 4 In L. lactis, most proteins are secreted unfolded via the secretion (Sec) pathway (13). Proteins are 56 synthesized as precursors containing the mature moiety of the protein with an N-terminal signal 57 peptide. The signal peptide plays an important role in targeting the protein to the cytoplasmic 58 membrane, where the protein precursor is subsequently translocated by the Sec machinery (14). 59 Following cleavage of the signal peptide, the mature protein is released extracellularly (15). The 60 lactococcal signal peptides follow the common tripartite structure including a positively charged 61 N terminus (n-region), a central hydrophobic core (h-region), and a more polar C terminus (c-62 region) containing the signal peptide cleavage site (16). The most widely used signal peptide for 63
An optimized system for expression and purification of secreted bacterial proteins
Protein Expression and Purification, 2006
In this report, we describe an optimized system for the eYcient overexpression, puriWcation, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-cmyc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, puriWed under denaturing conditions by immobilized metal aYnity chromatography, and refolded by rapid dilution into a solution containing only Tris buVer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further puriWcation and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further puriWed, analytical gel Wltration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-deWned secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins puriWed using this meth