Abstract
Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DI15 alpha strain of E. coli. The soluble and active enzyme was produced in E. coli 11121 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited a molecular weight of 29,200.94 Da by MALDI-TOF analysis. The purified enzyme showed specific activity of 1.64 U/mg with methionylp-nitroanilide and 1.51 U/mg with synthetic tetrapeptide substrate 'MGMM' in a discontinuous IIPLC-based assay. In vitro studies showed the processing of up to 36% of Met-INF alpha-2b in 40 min. In silico studies revealed that the ES-complex formation between the enzyme and interferon has a Delta G - 683.07 kJ/mol. Molecular docking results showed that the processed INF alpha-2b has greater binding affinity with IFNAR2 receptor as indicated by Delta G - 784.53 kJ/mol, significantly lower than that of methionine containing INF alpha-2b (Delta G - 717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processed recombinant interferon.