Cultivation at 6–10 °C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli

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Abstract

Protein expression in Escherichia coli at 15–25 °C is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 °C without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 l culture grown at 6 and 10 °C, respectively. Cultivation at 10 °C also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21(DE3). Consequently, E. coli cultivation at 6–10 °C is an effective strategy for overcoming a major hurdle of the inclusion body formation.

Highlights

► Escherichia coli was cultivated at 6–10 °C to produce recombinant proteins. ► E. coli strains without cold adapted chaperones grew at the extremely low temperatures. ► The active soluble proteins were produced in large quantity under this condition. ► This strategy is effective for overcoming the insolubility of recombinant proteins.

Introduction

Proteins play critical roles in the fundamental processes of life. Biochemical, biophysical, and structural information on proteins help to understand their biological functions. One major limitation to physicochemical analyses is the difficulty in preparing highly purified soluble proteins. Various protein expression systems have been developed for overproduction of recombinant proteins using diverse host organisms such as bacteria [1], yeasts [2], insects [3], and mammalian cells [4]. Among them, Escherichia coli has been preferred due to its fast growth, convenience in manipulation and low cost for protein production. On the other hand, recombinant proteins expressed in E. coli often faces solubility problem; that is, expressed proteins form inclusion bodies [5].

Protein expression at low temperatures sometimes prevents the formation of inclusion bodies [6]. Decreased translation rate at low temperatures seems to render a sufficient time for nascent polypeptides to fold correctly [7]. Protein expression at low temperatures could also alleviate heat denaturation of recombinant proteins and prevent degradation by heat shock proteases [8]. For these reasons, the sub-optimal temperatures between 15 and 30 °C are routinely employed for the production of recombinant proteins. It should be noticed, however, that many recombinant proteins form inclusion bodies when they are expressed in E. coli even at the sub-optimal temperatures.

In general, lowering the cultivation temperature under 15 °C has not been applied to E. coli expression systems. It is known that cultivation at ∼10 °C invokes severe consequences in E. coli physiology, including a decrease in the saturation of fatty acids and the inhibition of DNA, RNA, and protein synthesis [9], [10]. Nevertheless, E. coli can still grow at 7.5–7.8 °C, although the growth rate is drastically decreased [11]. For protein production at these low temperatures, E. coli ArcticExpress strains with cold-adapted chaperonins from a psychrophilic bacterium, Oleispira antarctica [12] have been developed. These strains can be used for protein production at 10 °C or lower temperatures. However, there are only a few examples of successful expression of recombinant proteins using the ArcticExpress strains [13], [14], [15]. In this study, we report the production of active recombinant proteins in the generally-used E. coli expression systems without cold adapted chaperones by simply lowering cultivation temperature down to the minimal growth condition.

Section snippets

Target proteins, plasmids, and bacterial strains

Proteins, plasmids, and bacterial strains used in this study are listed in Table 1. All proteins were expressed with C-terminal His6 tags. E. coli BL21(DE3) was used for the production of EM3L7, while E. coli Rosetta(DE3), Rosetta-gami2(DE3) and ArcticExpress(DE3)RIL were used for eukaryotic proteins, CaMan1 and

Production of soluble CaMan and CaCel in E. coli Rosetta-gami2(DE3) strain at 10 °C

After 3-day induction at 10 °C, the expression of CaMan and CaCel was examined in three different E. coli strains, Rosetta(DE3), ArcticExpress(DE3)RIL, and Rosetta-gami2(DE3). The crude lysate was separated into soluble and insoluble fractions by centrifugation and both fractions were analyzed by SDS–PAGE. Thick protein bands were observed at an expected position of CaMan in all the three insoluble fractions whereas expected protein bands of CaCel were observed in two insoluble fractions of

Discussion

In this study, we have demonstrated that E. coli cultivation at 6–10 °C is an effective strategy to overcome the insolubility problem of recombinant proteins. All the investigated proteins, which formed inclusion bodies when overexpressed at 15–37 °C, were found to be expressed as soluble forms in E. coli by simply lowering temperature under 10 °C. Although the reason for the soluble production of recombinant proteins under 10 °C is not clear, it would appear to be related to the reduced growth

Acknowledgments

We are grateful to Mr. Jung Ho Jeon of Korea Ocean Research and Development Institute (Korea) for contributing the pET-EM3L7 expression vector. This work was supported by the grant (PP00740) from the Korea Ocean Research and Development Institute and by the Marine & Extreme Genome Research Center Program from the Ministry of Land, Transport, and Maritime Affairs.

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