Bioprocessutveckling för produktion av rekombinant mussel-havstulpan protein i E. coli

The demand for biocompatible adhesives in the pharmaceutical industry has necessitated the development for sustainable alternatives to synthetic glues. This thesis investigates a novel bioprocess development and optimization for the recombinant production of an original, modular fusion protein inspired by the wet-adhesion mechanisms of marine mussels and barnacles. The target protein, characterized by intrinsically disordered domains and high positive charge, was expressed in Escherichia coli (E. coli) BL21-AI. To maximize product formation, upstream cultivations were scaled from 1.5 L to 5 L bioreactors under both batch and fed-batch modes. Critical process parameters, including induction timing, temperature regulation, and oxygen transfer rate (OTR), were modulated to enhance target protein expression while mitigating overflow metabolism and restricting anaerobic respiration. Downstream recovery was evaluated utilizing high-pressure homogenization (HPH), sonication, and immobilized metal affinity chromatography (IMAC). Results demonstrate that successful batch production requires induction prior to complete dissolved oxygen (DO) depletion. Furthermore, while fed-batch processing failed to yield the recombinant protein due to metabolic bottlenecks, the implementation of intermittent temperature regulation successfully restored product formation. Through optimization, yield of 10.0 mg recombinant protein/g substrate, volumetric productivity of 126.5 mg recombinant protein/ L culture media, and specific productivity of 0.19 recombinant protein mg/mL / mg/mL biomass were achieved. Finally, addressing identified oxygen transfer limitations in the current equipment, future work should explore pure oxygen supplementation to support high-density, multi-modular fusion protein production in E. coli.