High Level Expression and Purification of Recombinant Flounder Growth Hormone in E. coli

Abstract

In aquaculture, growth rate is one of the most important traits. Growing bigger fish in a shorter period of time is vital for the sustainability of aquaculture industry. This can be achieved by the use of recombinant growth hormones since native source is limited in availability. This study overproduced recombinant flounder growth hormone in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The synthetic flounder growth hormone gene was cloned in to PET-28a expression vector and transformed in to E. coli BL21(DE3). Induction at lower temperature, lower IPTG concentration and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. The attempt to produce soluble protein by reducing IPTG concentration, lowering induction temperature and co-expressing with chaperone did not yield soluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis with sonication and lysozyme treatment. Among the solublization buffers examined, buffer with 1 % N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solublization and recovery. The denaturant, N-lauroylsarcosine, was easily removed by filtration and dialysis. From 1 L culture of Terrific broth, 7.3 g wet weight of cell pellet was harvested and 1.79 g wet weight of inclusion bodies was obtained. Approximately 450 mg of purified recombinant flounder growth hormone was recovered from 1 L culture, which is significantly higher than previous reports that expressed several native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This expression and purification approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli.