An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.