Expression,purification and bioinformatics analysis of GroEL1 protein in mycobacterium tuberculosis
Objective:To study the prokaryotic expression and purification of GroEL1 from mycobacterium tuberculosis by ge-netic engineering and bioinformatics,and to predict its structure and function,and to analyze its application value in novel tubercu-losis vaccine.Methods:The GroEL1 gene was amplified by PCR in vitro and cloned into the pET28a plasmid.The pET28a-Gro-EL1 vector was successfully constructed by sequencing and transformed into the expression strain of E.coli BL21(DE3).The re-combinant GroEL1 protein was expressed by IPTG and purified by nickel affinity column.The nucleotide and amino acid sequences of GroEL1 of H37Ra strain were obtained from UniProt database.Protparam,TMHMM-2.0,Protscale,NetChop-3.1,Psortb and SignalP-4.1 were used to predict the physicochemical properties,transmembrane helix,hydrophilic/hydrophobic,phospho-rylation sites,subcellular localization and signal peptide of GroEL1 protein,respectively.NetNGlyc-1.0 and YinOYang-1.2 predicted the glycosylation sites.SOPMA and Swissmodel predicted the secondary structure and tertiary structure of the protein.Clustalw compares homologous sequences.String predicted interacting proteins,IEBD and ABCpred predicted B-cell epitopes of proteins.Results:The recombinant pET28a-GroEL1 vector was successfully constructed and the GroEL1 protein partially ex-pressed in soluble form in E.coli.The recombinant GroEL1 protein was purified by nickel affinity chromatography with a purity of more than 90%.Western blot analysis confirmed that the recombinant GroEL1 protein had good immunoreactivity.The GroEL1 gene of mycobacterium tuberculosis strain H37Ra has a total length of 1620 bp,encoding 539 amino acids with a molecular weight of 55.88 kD and an isoelectric point of 4.98.It is predicted that the protein has strong hydrophilicity,stable properties,no trans-membrane region,37 possible phosphorylation modification sites and 8 O-glycosylation sites,which is a non-secreted protein.Located in the cytoplasm,the secondary structure showed α-helix(53.43%),extended chain(11.87%),β-turn(7.61%),random coil(27.09%),multiple B cell antigen epitopes and multiple GroEL1 protein interacting proteins.Conclusion:The recom-binant GroEL1 protein was successfully expressed and purified,and the structure and function of GroEL1 protein were predicted by bioinformatics,which laid a foundation for the prevention,diagnosis and treatment of tuberculosis.