Membrane Vesicles Produced by Shewanella vesiculosa HM13 as a Prospective Platform for Secretory Production of Heterologous Proteins at Low Temperatures

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

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Expression of Heterologous Antigens in Commensal Neisseria spp.: Preservation of Conformational Epitopes with Vaccine Potential

Infection and Immunity ◽

10.1128/iai.72.11.6511-6518.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6511-6518 ◽

Cited By ~ 29

Author(s):

Clíona A. O'Dwyer ◽

Karen Reddin ◽

Denis Martin ◽

Stephen C. Taylor ◽

Andrew R. Gorringe ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Meningococcal Disease ◽

Protective Efficacy ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Heterologous Proteins ◽

Protein Vaccine ◽

Native Conformation ◽

Protein Antigens

ABSTRACT Commensal neisseriae share with Neisseria meningitidis (meningococcus) a tendency towards overproduction of the bacterial outer envelope, leading to the formation and release during growth of outer membrane vesicles (OMVs). OMVs from both meningococci and commensal neisseriae have shown promise as vaccines to protect against meningococcal disease. We report here the successful expression at high levels of heterologous proteins in commensal neisseriae and the display, in its native conformation, of one meningococcal outer membrane protein vaccine candidate, NspA, in OMVs prepared from such a recombinant Neisseria flavescens strain. These NspA-containing OMVs conferred protection against otherwise lethal intraperitoneal challenge of mice with N. meningitidis serogroup B, and sera raised against them mediated opsonophagocytosis of meningococcal strains expressing this antigen. This development promises to facilitate the design of novel vaccines containing membrane protein antigens that are otherwise difficult to present in native conformation that provide cross-protective efficacy in the prevention of meningococcal disease.

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In silico analysis of different signal peptides to discover a panel of appropriate signal peptides for secretory production of Interferon-beta 1b in Escherichia coli

Acta Biochimica Polonica ◽

10.18388/abp.2018_2351 ◽

2018 ◽

Author(s):

Shahrokh Ghovvati ◽

Zahra Pezeshkian ◽

Seyed Ziaeddin Mirhoseini

Keyword(s):

Escherichia Coli ◽

Interferon Beta ◽

Biologically Active ◽

Experimental Investigations ◽

Signal Peptides ◽

Cleavage Sites ◽

Heterologous Proteins ◽

Periplasmic Space ◽

E Coli ◽

Secretory Production

Signal peptides (SPs) are one of the most important factors for suitable secretion of the recombinant heterologous proteins in Escherichia coli (E. coli). The objective of this study was to identify a panel of signal peptides (among the 90 biologically active SPs) required for the secretory production of interferon-beta 1b (IFN-beta 1b) recombinant protein into the periplasmic space of E. coli host. In the initial step, after predicting the accurate locations of the cleavage sites of signal peptides and their discrimination scores using SignalP 4.1 server, 31 SPs were eliminated from further analysis because their discrimination scores were less than 0.5 or their cleavage sites were inappropriately located. Therefore, only 59 SPs could be theoretically applied to secrete IFN-beta 1b into the periplasmic space of E. coli. The physico-chemical and the solubility properties, which are necessary parameters for selecting appropriate SPs, were predicted using ProtParam and SOLpro servers using the 59 remaining signal peptides. The final subcellular localization of IFN-beta 1b in combination with different SPs was predicted using ProtComB server. Consequently, according to the ranking of 59 confirmed SPs, the obtained results revealed that SPs Flagellar P-ring protein (flgI), Glucan1,3-beta-glucosidase I/II (EXG1) and outer membrane protein C (OmpC) were theoretically the most potentand desirable SPs for secretion of recombinant IFN-beta 1b into the periplasmic space of E. coli. For further studies in the future, the experimental investigations on the obtained results will be considered.

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Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation

Biochemical Journal ◽

10.1042/bj3060057 ◽

1995 ◽

Vol 306 (1) ◽

pp. 57-61 ◽

Cited By ~ 6

Author(s):

M A Kaderbhai ◽

V J Harding ◽

A Karim ◽

B M Austen ◽

N N Kaderbhai

Keyword(s):

Protein Translocation ◽

Membrane Vesicles ◽

Heterologous Proteins

A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep. These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes.

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Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

Applied and Environmental Microbiology ◽

10.1128/aem.07209-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 651-659 ◽

Cited By ~ 18

Author(s):

Liuyang Diao ◽

Qilei Dong ◽

Zhaohui Xu ◽

Sheng Yang ◽

Jiahai Zhou ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Targeting ◽

Heterologous Proteins ◽

Gram Positive ◽

Content Type ◽

E Coli ◽

New Strategy ◽

Secretory Production ◽

Close Relatives

ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

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