Bioprocess development for therapeutic proteins and enzymes

• Post by: wangikar_8khp2z
• 11:01AM Apr 11, 2018
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The lab has developed fed-batch, high cell density cultivation protocols for the production of soluble proteins in E. coli BL21 (DE3). This fed-batch strategy is based on the reported high cell density protocols for E. coli with modifications to significantly improve soluble protein production.

Representative profile for the dissolved oxygen and substrate feed in a fed-batch process.

Our strategy uses minimal medium that reduces the raw material costs while providing a density of 100 gm dry cell weight/liter. Our novel control loops ensure growth under carbon limiting conditions thereby minimizing overflow metabolism, acetate formation and repression of protein expression. For example, Lactobacillus kefir alcohol dehydrogenase was produced at 7.1 X 106 units/L corresponding to about 12.9 gm soluble protein/L. The strategy has been demonstrated for a number of proteins and enzymes. Additionally, researchers in the lab have fine-tuned strategies for the production of Alcaligenes faecalis -nitrilases and Candida boidinii – formate dehydrogenase enzymes and a number of proteins of therapeutic interest.

Broader Impact- Expression systems based on a strong promoter such as T7 promoter and an operator such as the lac operator are routinely used and work quite well at the laboratory level. Characteristics of such expression systems that are relevant to a production process are: (i) Excess carbon substrate and high growth rates lead to an overflow metabolism resulting in accumulation of products such as acetate, which then adversely affect growth and protein production; (ii) Protein expression remains repressed when carbon substrates such as glucose are abundant and (iii) addition of an inducer leads to expression of protein at high levels and possible formation of inclusion bodies. These characteristics make commercial level protein production a challenge. Our process not only achieves higher biomass but also proteins with higher solubility thereby eliminating the need for the lengthy procedure of protein refolding. The process has applications in the production of industrial enzymes, therapeutic proteins and bulk chemicals.