Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Abstract

BackgroundSilk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.ResultsThis study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.ConclusionsWe have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

“…Around 154 mg of the fusion protein was purified from a 12-L fermentation culture and the yield of ELP was$1.8 mg/L after cleavage by factor Xa and purification. Finally, higher levels of ELP20 production could be foreseen after extensive optimization of the process, as was recently described for the production of silk-elastin-like polymers [33][34][35].…”

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

“…In an attempt to enhance the commercial viability of these novel polymers we undertook a project aimed at increasing SELP productivity and developing a scalable cost effective production process for these2122. Our initial study focused on optimising batch production in the E. coli BL21(DE3)-pET expression system via an in-depth empirical approach comparing all process variables and enabled an increase in productivity up to 500 mg/L purified SELP21.…”

“…In an attempt to enhance the commercial viability of these novel polymers we undertook a project aimed at increasing SELP productivity and developing a scalable cost effective production process for these2122. Our initial study focused on optimising batch production in the E. coli BL21(DE3)-pET expression system via an in-depth empirical approach comparing all process variables and enabled an increase in productivity up to 500 mg/L purified SELP21. In addition, factors potentially limiting further improved productivities were identified and included an accumulation of the metabolic byproduct acetate to toxic levels, plasmid instability of culturable cells and increased host cell metabolic burden following induction of production21.…”

“…Our initial study focused on optimising batch production in the E. coli BL21(DE3)-pET expression system via an in-depth empirical approach comparing all process variables and enabled an increase in productivity up to 500 mg/L purified SELP21. In addition, factors potentially limiting further improved productivities were identified and included an accumulation of the metabolic byproduct acetate to toxic levels, plasmid instability of culturable cells and increased host cell metabolic burden following induction of production21. Subsequently, we circumvented the former limitation by developing and optimising a scalable fed-batch process which reduced acetate accumulation and increased SELP yield to 4.3 g L −1 , the highest reported value for any PBP to date22.…”

Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L.) in three E. coli expression strains

“…MBP-and His-tagged CBF1 protein was purified onto amylase and nickel column, respectively. Batch production of heterologous protein in E. coli BL21-CodonPlus® (DE3)-RIPL was performed by Collins et al (2013). Maximise production of the silk-elastin-like protein involved optimalisation of media, induction time and temperature.…”