Dealing with purification challenges - IMAC Series, Challenge Two

Sep 26 2017 0 Comments Tags: Affinity purification, His-tagged protein purification, IMAC resins, Ni-Penta resins, Protein function, Protein structure, recombinant protein production

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 Two: Refolding of protein from E. coli inclusion bodies


The challenge: The protein segregated into the inclusion bodies needs to be denatured, purified and then refolded, requiring a large volume of buffer changes.

          The process is very time-consuming, expensive, and poorly scalable as large quantities of reagents are being used. Also, it is challenging to determine refolding conditions for each specific protein.

The solution: On-column refolding

              We have previously discussed that high level expression of recombinant protein in Escherichia coli often results in aggregation of the expressed protein molecules into inclusion bodies. Modulating the expression rate of the target protein, by using a weaker promoter for transcription, lowering inducer concentration, or lowering incubation temperature during expression, often helps to improve its solubility in E.coli (Read more on this). However some proteins possess such amino acid compositions and properties that determine their insoluble expression in the inclusion bodies inevitably. These proteins must be isolated from the inclusions bodies. Solubilization of Inclusion bodies (IB) and protein refolding - hopefully to its native conformation - has been proposed as another purification challenge. The protocol involves several factors to consider and parameters to empirically determine to avoid degradation and aggregation of the target protein during the process. It is a lengthy, tedious procedure with a lot of liquid handling, referred as “a commonly tried out but only episodically successful protocol” [1].

             Let’s discuss together how to tackle this challenge. First, we need to remind ourselves the inclusion bodies are not all bad after all. Inclusion bodies contain very little host protein, ribosomal components or DNA/RNA fragments. They often contain mostly the over expressed protein. Their high buoyant density facilitates their separation from soluble E. coli proteins and cell debris by differential centrifugation or few sequential centrifugation steps. Very often, the purity of target proteins isolated from the inclusion bodies is satisfactory. Importantly, aggregation in inclusion bodies are reversible!

              In most common applications, the inclusion bodies will be solubilized by a strong denaturant, such as 6 M GuHCl or 8 M urea. Then the target protein with a fusion tag will be purified on the chromatography column, such as IMAC for the His tag. The column will be washed several times to remove impurities. Sequentially, the target protein will be eluted from the column with imidazole in the denaturant. After elution, protein refolding will be achieved by depletion of the denaturant, frequently via dialysis against a large volume of buffer [2,3]. Either one-step or multi-stepped dialysis against a series decreasing gradient of the denaturant has been applied successfully to refold various proteins [2,3].

              Here, we are going to discuss an alternative approach to obtain the renatured target protein: refolding on column. In this protocol, the target protein will be refolded while it is bound on the IMAC column. At elution, the protein can be obtained with a non-denaturing eluant. It is mainly soluble and folded, while the misfolded counterparts that may remain aggregated will be mainly trapped on the column. Since the target protein is concentrated, bound on the column throughout renaturation, there are no liters-after-liters of dialysis buffer changes required. The advantages of this protocol are time-efficient, less liquid handling, and fully adaptable to automated systems for scaling up and screening.

              Oganesyan et al. demonstrated that the column-based refolding protocol could efficiently shorten a three-day dilution-based protocol to only 20 hours, removing the need of using large volume of dialysis buffers as well as post-dialysis concentration of the diluted sample [4]. Among the 10 proteins they attempted to refold on-column, five proteins could be refolded to 100% completion, 2 of which were successfully crystallized for solving protein structures [4]. Several other studies also reported that their target His tagged proteins could be successful purified and refolded from the inclusion bodies, including extracellular proteins and membrane proteins [5-7].

             On-column refolding is a very straight forward protocol. The following flow chart highlights the steps that are involved:

    On colume refolding

              Several specific parameters in the protocol that are protein dependent may need to be determined empirically. Here are some of the factors to be considered:

    1. The recommended renaturation buffer to start with is 20 mM Tris-HCl, pH 7.5, 0.1 NaCl. The pH of the buffer should be adjusted to be at least 1.0 pH units away from the pI of the protein to avoid protein precipitation [3]. Denaturant gradients can be included in the buffer. For some proteins, a denaturant concentration may also need to be determined to populate the target protein at the intermediate state during folding transition.
    2. Reducing reagents, such as b-ME and DTT, may be included in the refolding buffer to avoid dimerization and aggregation if the amino acid sequence of the target protein contains cysteine residues.
    3. Additives can be added to the system to facilitate the renaturation process. Mild detergents (such as Triton-100 and NP-40) and Arginine have been used as aggregation suppressor [2]. Molecular chaperons, such as b-cyclodextrins, have been successfully used as folding enhancers [3,8].

              On a further note, refolding on-column is compatible with automated purification. When performed on an automated system, refolding procedure can be completed in 60 min to 120 min with a linear decreasing denaturant gradient at a slow flow rate (for example, 0.5 mL/min). Automation of the on-column renaturation process has also enabled rapid high-throughput screening of refolding conditions for target proteins [9,10].



    1. Structural Genomics Consortium et al. Protein production and purification. Nat Methods. 2008 Feb; 5(2): 135–146.
    2. Tsumoto K, et al. Practical considerations in refolding proteins from inclusion bodies. Protein Expression and Purification 28 (2003) 1–8
    3. Yamaguchi H and Miyazaki M. Refolding Techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules. 2014 Mar; 4(1): 235–251.
    4. Oganesyan N, et al., On-column protein refolding for crystallization. J Struct Funct Genomics. 2005;6(2-3):177-82.
    5. Zhu X et al., On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli. 2005 Acta Biochimica et Biophysica Sinica, 37(4): 265–269
    6. Thomson C et al., A simplified method for the efficient refolding and purification of recombinant human GM-CSF. 2012 PLoS One 7(11): e49891.
    7. Zhai L et al., A rapid method for refolding cell surface receptors and ligands. Scientific Reports 2016 24;6:26482.
    8. Sasaki Y, Akiyoshi K. Development of an artificial chaperone system based on cyclodextrin. Curr Pharm Biotechnol. 2010 Apr;11(3):300-5.
    9. Qoronfleh MW et al., Confronting high-throughput protein refolding using high pressure and solution screens. Protein Expr Purif. 2007 Oct;55(2):209-24.
    10. Cothran A et al., A medium or high throughput protein refolding assay. Biotechnol Prog. 2011 Sep-Oct;27(5):1273-81.


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