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A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

ABSTRACT: Lactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

SUBMITTER: Lim PY

PROVIDER: S-EPMC5715515 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

Lim Pei Yu PY Tan Lee Ling LL Ow Dave Siak-Wei DS Wong Fong T FT

Microbial cell factories 20171205 1

<h4>Background</h4>Lactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases ...[more]

PMID: 29207979

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Project description:BackgroundLactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development.ResultsA recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development.ConclusionsIn this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.

Dataset Information

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

Publications

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

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