Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Abstract

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (P _{T7 lac} , P_trc, P_tac, and P_BAD) and two different replication origins (high copy number pMB1' and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

Keywords: Escherichia coli; expression system; microbial factory; origin of replication; promoter; recombinant protein.

FIGURE 1

Scheme of expression vectors constructed in this study. PMCSR, promoter multicloning site region; KSD, Kozak Shine-Dalgarno; YFP, yellow fluorescent protein; Amp^R, ampicillin resistance.

FIGURE 2

Percentage of yellow fluorescent protein (YFP) expression with respect to maximal expression for each plasmid under different inductor conditions. The YFP expressions selected were the highest achieved at stationary growth phase. (A) E. coli wt growing with glucose as carbon source. (B) E. coli wt growing with glycerol as carbon source. (C) E. coli ΔackA growing with glucose as carbon source. (D) E. coli ΔackA growing with glycerol as carbon source.

FIGURE 3

Dependence of inductor on YFP expression time. (A) YFP expression of E. coli wt carrying the pSF-pMB1’-tac-YFP and growing with glucose as carbon source at different IPTG concentrations. Yellow arrow shows induction time with 0.1 mM IPTG. (B) YFP expression time (in hours after 0.1 mM IPTG addition) of E. coli wt carrying the different lac plasmids constructed growing in TB7 supplemented with glucose as carbon source at different IPTG concentrations. (C) YFP expression time (in hours after 2 mM L-arabinose addition) of E. coli wt carrying the P_BAD plasmids constructed growing in TB7 supplemented with glucose as carbon source at different L-arabinose concentrations. (D) E. coli wt carrying pSF-pMB1′-BAD-YFP growing with glucose as carbon source. (E) E. coli wt carrying pSF-pMB1′-BAD-YFP growing with glycerol as carbon source. Green dots show grown at 600 nm, while black dots show YFP fluorescence in arbitrary units (RFU). Gray square corresponds with culture growth, and pink square indicates YFP expression. Purple square indicates hours when culture is growing and expressing YFP simultaneously. Yellow arrow shows 2 mM L-arabinose induction time.

FIGURE 4

YFP expression of E. coli wt or deficient ackA mutant growing with glucose or glycerol as carbon sources carrying different expression system vectors. Expression is shown as a percentage with respect to expression of E. coli wt transformed with the plasmid pSF-p15A-trc-YFP growing with glycerol as the 100%. Secondary y-axis shows YFP concentration (mg/L). Expression of E. coli wt is shown in orange and E. coliΔackA in green. Glucose cultures are in solid bars, while glycerol supplemented cultures are shown in striped bars.

FIGURE 5

Soluble and insoluble percentage of YFP measured for each expression vector. The fluorescence is indicated as a percentage, with 100% as observed with the pSF-p15A-trc-YFP vector. Carbon source, host strain, strength of the promoter, inductor concentration, and vector copies are shown under the graph. Statistical analysis by one way ANOVA was carried out with Graphpad Prism 7.0 [p value < 0.0001 (^****), <0.001 (^∗∗∗), <0.01 (^∗∗), and <0.05(^∗)].