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| Expression of Drug-resistant Genes in Streptococcus pneumoniae and Purification and Functional Analysis of Its Protein |
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Abstract Abstract Streptococcus pneumoniae is one of the major human (Homo sapiens) pathogens and leads to approximately 1.6 million deaths annually in the world. Owing to the abuse of the antibiotics, S. pneumoniae generated antibiotic resistance, especially the multiresistance to many antibiotics. Thus it is urgent to develop new antibiotics and vaccines. Based on bioinformatics analysis，a gene SP_0010 (GenBank No.: A0A0H2UMY6-1) was identified from S. pneumoniae strain TIGR4 that encodes a putative β-lactamase. In this study, the potential β-lactamase gene SP_0010 was amplified by polymerase chain reaction and inserted into the pET28a(+) vector to obtain the recombinant expression plasmid, which was then transformed into Escherichia coli strain Rosetta (DE3) for expression. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the optimum induction temperature of the recombinant protein Sp_0010 was 16 ?C for 20 h, meanwhile, the target protein was eluted with 250 mmol/L imidazole from Ni-NTA and loaded onto a Superdex-200 column for further purification. The recombinant Sp_0010 existed as a monomer in solution and has high purity. Peptidoglycan binding assay revealed that the recombinant Sp_0010 showed a stronger binding affinity to teichoic acid-free peptidoglycan. All these findings revealed that protein Sp_0010 had an important role in peptidoglycan metabolism. Thus it would have great significance to elucidate the mechanism of pneumococcal antibiotics resistance.
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Received: 22 January 2018
Published: 20 July 2018
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