Expression,Purification And Bioinformatics Analysis Of Mycoplasma Amphoriforme P1 Protein

ObjectiveTo find out the P1 adhesion-like virulence factor in Mycoplasma amphoriforme than the same of P1 adhesion in Mycoplasma pneumoniae by bioinformatics analysis,and then to clone and prokaryotic express this protein.Methods1.Bioinformatics analysis of P1 adhesion-like in Mycoplasma amphoriforme(named Mycoplasma amphoriforme P1).The position of Mycoplasma pneumoniae P1 adhesion in Mycoplasma pneumoniae genome and amino acid sequence were used as templates to screen the most similar Mycoplasma amphoriforme P1 from Mycoplasma amphoriforme genome.Through the online software,the bioinformatics analysis of Mycoplasma amphoriforme P1 was carried out,the physical and chemical properties were analyzed,and the signal peptide,transmembrane region,phosphorylation site,secondary structure,tertiary structure,domain and antigenic determinant were predicted.2.Recombinant expression and purification of MA-P1 protein.Based on the results of bioinformatics analysis,Mycoplasma amphoriforme P1 was trunked and optimized(named MA-P1).The optimized MA-P1 gene was synthesized and subcloned into the prokaryotic expression vector p ET-30 a.The recombinant plasmid p ET-30aMA-P1 was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG for expression.Finally,the target protein was purified by Ni-chelating affinity chromatography.The expression products were analyzed through SDS-PAGE and Western-blot.Results1.The protein was encoded by gene 184359-187322 of Mycoplasma amphoriforme(protein ID was CDN40264.1),which had the highest position and amino acid sequence similarity with P1 adhesion in Mycoplasma pneumoniae,so it was named as Mycoplasma amphoriforme P1.Mycoplasma amphoriforme P1 gene encoded 987 AA,the molecular weight was about 107 k Da,the Theoretical p I was 9.27,the Instability index was 29.59(< 40),and grand average of hydropathicity was-0.219.The probability of the presence of signal peptide was 24.64%.The protein formed a transmembrane region between amino acids at positions 13-35 and 882-904,respectively.There were46 serine phosphorylation sites,19 threonine phosphorylation sites and 6 tyrosine phosphorylation sites.The secondary structure consisted of α helix,extended chain,βAngle and random coil,and the proportions of each structure were 19.66%,23.91%,8.51% and 47.92%.A low complexity domain was formed between the amino acids at660-668,716-725 and 958-971,and a protein domain may be formed between the amino acids at 96-151.There were 33 epitopes,and the average propensity was 1.0160.2.Based on the results of bioinformatics analysis,amino acids at sites 1-313 of Mycoplasma amphoriforme P1 were truncated as the target protein,which was named MA-P1.MA-P1 gene was synthesized and subcloned into the prokaryotic expression vector p ET-30 a.The recombinant plasmid was transferred into E.coli BL21(DE3)and induced by IPTG for expression.SDS-PAGE and Western-blot analysis showed that the molecular weight of MA-P1 was about 35 k D,which was consistent with the expected molecular weight of the target protein.MA-P1 was purified by Ni-chelating affinity chromatography,and the final concentration of the target protein was 1.2 mg/m L.Conclusions1.Mycoplasma amphoriforme P1 was the protein encoded by gene 184359-187322 of Mycoplasma amphoriforme,and the protein ID was CDN40264.1,which was similar to the position and amino acid sequence of Mycoplasma pneumoniae P1 adhesion.2.Mycoplasma Amphoriforme P1 was analyzed by online biological software.Based on the analysis results,it was trunked and preliminarily optimized,and named MA-P1.3.The recombinant vector p ET-30a-MA-P1 was successfully constructed and could be expressed efficiently in the expression system of E.coli.The target protein with high purity was obtained by Ni-chelating affinity chromatography,which provided the raw material basis for the establishment of Mycoplasma amphoriforme ELISA method in the later stage.