Objective:To study the prokaryotic expression and purification of GroEL1 from mycobacterium tuberculosis by genetic engineering and bioinformatics, and to predict its structure and function, and to analyze its application value in novel tuberculosis vaccine. Methods:The GroEL1 gene was amplified by PCR in vitro and cloned into the pET28a plasmid. The pET28a-GroEL1 vector was successfully constructed by sequencing and transformed into the expression strain of E. coli BL21 (DE3). The recombinant 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, phosphorylation 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 expressed 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 1 620 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 transmembrane 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 recombinant 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.
WEI Jing
,
GUO Fangzheng
,
SONG Yamin
,
LI Baiqing
,
XU Tao
,
WANG Hongtao
. Expression, purification and bioinformatics analysis ofGroEL1 protein in mycobacterium tuberculosis[J]. Journal of Baotou Medical College, 2024
, 40(7)
: 23
-31
.
DOI: 10.16833/j.cnki.jbmc.2024.07.004
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