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Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP.

ABSTRACT: Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

SUBMITTER: Park AR

PROVIDER: S-EPMC8578067 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHeterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter.ResultsThe YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions.ConclusionsThe new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.

Dataset Information

Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP.

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