Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b

Abstract

Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionineaminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes therecombinant production and properties of MAP from the DH5α strain of E. coli. The soluble and active enzyme was produced in E.coli BL21 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited amolecular 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 HPLC-based assay. In vitro studiesshowed the processing of up to 36% of Met-INFα-2b in 40 min. In silico studies revealed that the ES-complex formation between theenzyme and interferon has a ΔG –683.07 kJ/mol. Molecular docking results showed that the processed INFα-2b has greater bindingaffinity with IFNAR2 receptor as indicated by ΔG –784.53 kJ/mol, significantly lower than that of methionine containing INFα-2b(ΔG –717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processedrecombinant interferon.