IMAC for purification of poly Histinine tag is highly specific, yet inexpensive separation method to obtain highly pure protein. The poly Histinine tag is rather small and has very minimal impact on the conformation and function of the recombinant proteins. However, every protein is different. No one protocol fits all. There are many challenges that scientists have been encountering in purifying their proteins. At Marvelgent, we provide tool tips and tricks based on our knowledge and experience, and hope you will find them helpful in tackling the challenges.
Challenge One: Improving soluble expression of target protein
The challenge: Some His-tagged proteins and peptide fragments are prone to aggregate to form inclusion bodies when expressed in E.coli.
Production of these proteins requires several expensive, time-consuming steps as cell lysis, inclusion body solubilization, refolding, and concentration. Nonetheless, renaturation of the protein might not lead to fully native, bioactive conformation. The refolded protein might also be prone to aggregate when buffer condition changes.
The solution: Changing expression conditions to improve expression of a soluble protein.
- Lowering the temperature to slow down protein production. Low temperature decreases the rate of protein synthesis, facilitating proper folding of the protein and alleviating the toxicity of some proteins. Many times room temperature induction gives satisfactory results, whereas more controlled temperature at 15 or 20 Celsius degree is necessary to increase soluble expression of some proteins (1, 2).
- Using low concentrations of inducer. In general, for T7 promotor in many applications, a IPTG concentration within the range of 0.05 -2.0 mM will be used to induce the target protein expression. Lower IPTG concentrations help to produce soluble proteins for some targets (3). In one study, as low as 0.1 µM IPTG was found as the optimal IPTG concentration for obtaining maximal solubility of GST-PTEN. (4). However, studies on some other target proteins suggested that solubility is not necessarily associated with low IPTG concentration being used (2).
Changing E.coli strain. The strain or genetic background for recombinant expression is highly important. It has been reported that some E. coli strains may help optimize the level of soluble expression (2, 5, 6).
The BL21(DE3) and its derivatives are by far the most used strains for protein expression. Rosetta strains contain codons that are frequently present in eukaryotic proteins but rarely used in E.coli, therefore, the strains are very useful in the expression of eukaryotic proteins. For the target protein that contains disulfide bond, a host strain with a more oxidizing cytoplasmic environment with help improving its solubility. The Origami strain, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor), provides a favorable cellular conditions to facilitate proper disulfide bond formation and is commercially available.
- Select alternative microbial expression hosts. For example, recombinant human pro-relaxin H2 has been mostly purified from the inclusion bodies using a costly, time-consuming approach. A more cost-effective protocol was recently proposed, where a His-tagged version of pro-relaxin H2 was induced in Pichia pastoris. Being expressed in yeast, the recombinant protein was properly folded and secreted into the culture media, which could be readily purified by IMAC to high purity (7).
- Larentis AL et al., Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Res Notes. 2014 Sep 25;7:671.
- Hartinger D et al., Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1. Microb Cell Fact. 2010 Aug 18;9:62.
- Rabhi-Essafi I et al., A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli. Protein Eng Des Sel. 2007 May;20(5):201-9.
- Hu Y et al., The Optimization of Soluble PTEN Expression in Escherichia coli. Open Biochem J. 2015 Aug 21;9:42-8.
- Berrow NS et al., Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1218-26.
- Vernet E et al., Screening of genetic parameters for soluble protein expression in Escherichia coli. Protein Expr Purif. 2011 May;77(1):104-11.
- Cimini D et al., Production of human pro-relaxin H2 in the yeast Pichia pastoris. BMC Biotechnol. 2017 Jan 14;17(1):4.
To select the right type of IMAC media for the purification of your precious protein, read our suggestions:
Tech Note: IMAC Resins Selection Guide