Improving protein secretion by engineering components of the bacterial translocation machinery

Braun, Peter; Gerritse, Gijs; Dijl, van Jan Maarten; Quax, Wim J.

doi:10.1016/s0958-1669(99)80068-8

Cited by 38 publications

(25 citation statements)

References 31 publications

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“…About 55% of the identified clones could not be expressed in an FhuA-based surface display system in E. coli. Besides the possibility that toxic fragments interfere with growth, it is known that membrane permeation of certain protein domains is a challenge due to the high selectivity of the outer membrane translocase (1). Besides for bacterial surface display, membrane perme- ation is also required for (nonlytic) phage display (e.g., M13 based) as well as yeast display; our observation can therefore probably be generalized also to these systems, although the extent to which the generalization applies might vary.…”

Section: Discussionmentioning

confidence: 99%

Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

et al. 2003

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An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.

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Section: Discussionmentioning

confidence: 99%

Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

et al. 2003

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“…These eubacterial species are particularly attractive for this purpose because they have a high capacity to secrete proteins into the growth medium and because of their nonpathogenicity. Moreover, good fermentation technologies exist for various bacilli (36,40,41,263). Many proteins can be secreted to very high levels by B. subtilis, such as the ␣-amylase AmyQ from Bacillus amyloliquefaciens (1 to 3 g/liter) (204), protein A from Staphylococcus aureus (Ͼ1 g/liter) (91), and human interleukin-3 (227).…”

Section: Scope Of This Review-the Secretomementioning

confidence: 99%

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

Tjalsma

Bolhuis

Jongbloed

et al. 2000

Microbiol Mol Biol Rev

751

768

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SUMMARY One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major “cell factories” for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major “Sec” pathway for protein secretion. In contrast, the twin-arginine translocation “Tat” pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as “special-purpose” pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

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“…This was indeed established by the comparative immunization experiment, where the relevant strains containing the strong-promoter-pagA cassettes were evaluated in vivo (Table 3). Nevertheless, if, as suggested, the high level of PA expression introduces secretion stress, it is possible to use genetic manipulations, such as overexpression of the limiting factors involved in maturation and folding (8,24,47), and thus allow the generation of a stable vaccine strain or B. anthracis cells that could express and effectively secrete even higher levels of PA.…”

Section: Discussionmentioning

confidence: 99%

Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

Gat¹,

Inbar²,

Aloni-Grinstein³

et al. 2003

Infect Immun

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. To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the ␣-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cellassociated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

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Improving protein secretion by engineering components of the bacterial translocation machinery

Cited by 38 publications

References 31 publications

Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

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