Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

Abstract

The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high-level production of eukaryotic proteins. Two high-cell-density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207-234) recently and a high-cell-density IPTG-induction method described in this study, to achieve a cell-density OD(600) of 10-20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high-cell-density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14-25 mg of NMR triple-labeled proteins and 17-34 mg of unlabeled proteins from a 50-mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.

Figure 1

A schematic diagram of three expression methods used in this study.

Figure 2

A 12% SDS-PAGE of protein expression levels of human apoAI in D₂O. Lane 1: The traditional IPTG-induction method (0.4% ¹³C-glucose and 0.25 mM IPTG) at 20°C after IPTG induction. Lane 2: The high-cell-density IPTG-induction method (0.6% ¹³C-glucose and 0.25 mM IPTG). The cells were harvested at 12 h after IPTG-induction at 20°C. Lane 3: The autoinduction (0.4% ¹³C-glycerol) and the cells were harvested at 35 h at room temperature. MK—molecular weight marker. An arrow indicates the band position of human apoAI.

Figure 3

SDS-PAGEs of protein expression of apoE(1-215)/pTYB1 in D₂O before (Panel A), during (Panel B), and after (Panel C) double-colony selections. Arrows indicate the expected protein band (∼80 kDa, apoE(1-215) + intein + CBD). Panel A shows four different colonies before colony selection. Panel B shows results of three different colonies selected from the single-colony selection (Lanes 1–3) and another three colonies selected from the double-colony selection (Lanes 4–6). The second-colony selection was based on Colony 3 (Lane 3) in the single-colony selection, because this colony gave a higher protein production. Panel C shows the results of six colonies from the double-colony selection, indicating a high protein expression level of all six colonies. Molecular weight markers are labeled with kDa.

Figure 4

Left Panel: An SDS-PAGE showing autoinduction time course of triple-labeled human apoAI expression in D₂O at room temperature. The expected apoAI band is indicated by an arrow. Lane 1:24 h, Lane 2:28 h, Lane 3:32 h, Lane 4:36 h, Lane 5:40 h, Lane 6:44 h, and Lane 7:54 h. Panel B: Western blot of the same time course using an anti-human apoAI monoclonal antibody, 5F6.

Figure 5

12% SDS-PAGEs of autoinduction expression of human apoAI using the modified recipe of C-750501 medium containing different concentrations of glycerol. The glycerol concentrations are indicated in the bottom of the figure. For 0.3, 0.4, 0.5, and 0.75% glycerol, Lane 1:36 h, Lane 2:38 h, Lane 3:40 h, and Lane 4:43 h. For 0.2% glycerol, Lane 1:27 h, Lane 2:30 h, Lane 3:33 h, and Lane 4:36 h. MK: molecular weight marker.

Figure 6

A 12% SDS-PAGE of glucose optimization of human apoE expression in D₂O at 20°C using high-cell-density IPTG-induction bacterial expression: Uninduced (Lane 1), with 0.4% (Lane 2), 0.6% (Lane 3), 0.8% (Lane 4), and 1.0% glucose (Lane 5). Molecular weight marker is shown in left lane. Small-scale time course experiments with different glucose concentrations were also carried out to find the optimum protein expression time after induction of the culture.

Figure 7

¹H-¹⁵N HSQC spectra of triple-labeled human apoE(1-183) obtained using high-cell-density IPTG-induction expression (Panel C), autoinduction expression (Panel B), and traditional IPTG-induction expression (Panel A). All three samples contained 1.0 mM triple-labeled human apoE(1-183) in 100 mM phosphate buffer, 10 mM EDTA, 0.5 mM NaN₃, 90 mM DTT, and 0.02 mM DSS, pH 6.80. The spectra were collected at 30°C on a 600 MHz NMR spectrometer with a cold probe.