Alternative production process strategies in E. coli improving protein quality and downstream yields

Abstract

Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g −1 DCW, but the final specific enzyme activity (1.7 AU mg −1 RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield: (i) Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g −1 DCW. The enzyme activity increased to 4 AU mg −1 RhuA and purification-immobilization yield to 93%. (ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g −1 DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg −1 RhuA, and 95% of immobilization yield was achieved. For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.