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Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli
Sudip Ghosh1, 2, Sheeba Rasheedi2, Sheikh Showkat Rahim2, Sharmistha Banerjee2, Rakesh Kumar Choudhary2, Prachee Chakhaiyar2, Nasreen Z. Ehtesham1, Sangita Mukhopadhyay2, Seyed E. Hasnain2, 3
1, National Institute of Nutrition (ICMR), Hyderabad
2, CDFD, Nacharam, Hyderabad
3, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India
BioTechniques, Vol. 37, No. 3, September 2004, pp. 418–423
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The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.


Escherichia coli is the most widely used system for the rapid and economical production of recombinant proteins because of its very well-characterized genetics and rapid growth rate in inexpensive culture media. One major disadvantage of E. coli is that heterologous proteins are often expressed as insoluble aggregates of folding intermediates known as inclusion bodies. Expression in soluble fraction is paramount for the expressed protein to be biologically active. In order to recover soluble proteins from the inclusion bodies, the inclusion bodies are solubilized in the presence of strong denaturants such as urea or guanidinium hydrochloride, followed by the removal of the denaturants under optimal conditions that favor refolding. Although considerable progress has been made for efficient refolding of proteins (1), specific folding conditions differ greatly from protein to protein. Even under optimal conditions of refolding, quite a large number of proteins are found to be recalcitrant to refolding, and the yield of rena-tured protein is relatively low.

Several general and protein-specific methods are available for increased solubility of expressed proteins in E. coli. One approach is the coexpression of molecular chaperones, which assists in the correct folding of the heterologous protein (2,3). Similarly, concomitant overexpression of thioredoxin (TrxA) is known to improve the solubility of the expressed proteins (4). Another approach that has gained considerable success in recent years is the use of gene fusion (5). Fusion partners such as glutathione-S-transferase (GST) and maltose binding protein (MBP) are known to impart solubility of many heterologous proteins in addition to serving as a tag for affinity purification. Sometimes, soluble expression can also be enhanced by supplying essential cofactors necessary for the activity of the protein in question. For example, soluble expression of human cystathionine β-synthase, a heme-containing protein, could be increased over 8-fold by the addition of the heme precursor δ-aminolevulinic acid (δ-ALA) to the culture medium (6). In some instances, coexpression of nuclear receptor partners is also found to increase the solubility of nuclear receptors expressed in E. coli (7). In yet other cases, specific substitution of some amino acid residues was found to enhance the solubility of the expressed proteins (8). In this report, we describe a novel method of enhancing the solubility of expressed proteins by inducing recombinant protein expression in the presence of the dipeptide glycylglycine. We deliberately chose as an example mycobacterial proteins that are known to be difficult to express as soluble proteins in E. coli. The solubilization of these proteins was enhanced up to 170-fold.

Materials and Methods

Preparation of Recombinant Constructs

The open reading frames (ORFs) Rv0256c, Rv2430c (both are the members of the PPE gene family), Rv3339c (isocitrate dehydrogenase-1), and Rv1609 (anthranilate synthase) of Mycobacterium tuberculosis were amplified from the genomic DNA of the strain H37Rv by PCR. Oligodeoxyribonucleotide primers were chemically synthesized (Microsynth GmbH, Balgach, Switzerland), with appropriate restriction sites suitable for in-frame cloning into expression vectors, with N-terminal 6x-histidine tags. The primers used for amplification are shown in (Table 1). The recombinant proteins were expressed as N-terminal His-tagged fusions. The positive clones were confirmed by DNA sequencing.

Table 1. Sequence of the Primers, Restriction Sites, and Vectors Used for Expressing Different Mycobacterial Proteins

Restriction enzyme sites have been underlined.

Cell Culture

Competent BL21(DE3)pLysS cells (Novagen, Abingdon, UK) were transformed with pRSET256, pRSET1609, and pRSET3339 plasmid DNA, and the colonies were grown overnight on Luria Bertani (LB) plates (9) containing 100 µg/mL ampicillin. For pQE2430, competent M15(pREP4) cells (Qiagen GmbH, Hilden, Germany) were used and grown in LB plates containing ampicillin (100 µg/mL) and kanamycin (50 µg/mL). Fresh colonies were first inoculated into 5 mL LB media containing appropriate antibiotics and grown overnight at 37°C with shaking. These overnight cultures were diluted 10-fold into 10 mL Terrific Broth (TB) medium (9), containing different concentrations (0, 50, 200, 500, and 1000 mM) of glycylglycine (Amersham Biosciences, Buckinghamshire, UK) and further grown at 37°C in an orbitory shaker till the absorbance (A)600 = 1. The cultures were cooled to room temperature, and protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactoside (IPTG) for 14–16 h at 27°C.

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