Efficient refolding of recombinant reteplase expressed in Escherichia coli strains using response surface methodology
Introduction
Escherichia coli is widely used as a prokaryotic host for recombinant expression of proteins due to its rapid growth, low cultivation cost and well-known genetic structure [1]. In spite of these benefits, in many cases production of foreign proteins in E. coli leads to the formation of inactive proteins in the form of inclusion bodies (IB) [2]. Production of insoluble recombinant proteins as IBs is associated with several advantages such as high level purity of the target protein, increased resistance to proteolytic degradation, fast and simple extraction of the inclusion bodies from soluble proteins by a single centrifugation step [3,4]. At the same time, formation of inclusion bodies includes a severe bottleneck, which is the requirement of protein renaturation and refolding step [5].
Refolding is a selective strategy to recover native conformation of proteins from aggregated state. In this process, recombinant proteins are converted to the native conformation through partially folded intermediates [6]. IBs are solubilized usually in the presence of chemical denaturants like urea, guanidine hydrochloride (GdnHCL) or N-lauroylsarcosine due to their capability in decreasing the non-covalent interactions between protein molecules [7]. Then denatured proteins are refolded to the active and native form by removal of the chaotropic agents and aid of chemical and biological additives. The additives support the solubility and stability of the native proteins and significantly enhance the yield of refolding process. Chemical additives usually do not disturb subsequent purification steps and can be removed from the final product by plenty of methods [6,8]. Major chemical additives which are used in refolding procedures include sugars, surfactants, polymers and amino acids which are functionally grouped into three types of denaturants, protein stabilizers, and protein aggregation inhibitors [2,7]. The combination of these additives provides an optimum condition to obtain correct and active conformation of recombinant proteins in refolding process [3,9]. But a strategy for determining the most promising interactions between variables for optimal refolding is crucial.
Response surface methodology (RSM) is one of the most popular optimization methods used within recent years. RSM consists of a group of statistical and mathematical techniques for developing model, improving and optimizing parameters which can influence a desired response [10]. The main advantage of RSM is decreasing the number of experiments required to evaluate the effect of several selected variables and their interactions [4]. There are many reports which show suitability of RSM for optimization of protein production steps. Not only RSM is used for optimization of expression and fermentation steps [17,18] but also RSM has been remarkably used for optimization of refolding procedure. Lipase from a Pseudomonas sp., peroxidase from Lepidium draba, amylase from Bacillus megaterium and camelid single domain antibody are the examples of proteins expressed in E. coli as inclusion bodies and RSM is successfully used for optimization of in vitro refolding. In all these cases RSM could successfully optimize in vitro refolding with few experiments and higher efficiency and yield [[19], [20], [21], [22]].
Reteplase as a deleted variant of tissue plasminogen activator, is being used for the removal of thrombi from blood vessels. The protein is also expressed as IB in E. coli and its refolding is a challenge due to presence of nine disulfide bonds [17,18]. In this study, we have used RSM to increase the efficiency of refolding and to elucidate the interaction between chemical additives with minimum number of experiments. In order to improve reteplase refolding, additives including sugars (trehalose, mannitol, sorbitol, sucrose), amino acids (l-histidine, l-methionine, l-cysteine, l-proline, l-argenine), polyols (glycerol, PEG-1000) and surfactants (Tween-80, Tween-20, Triton X-100) were evaluated. At first, the effect of each chemical additive was investigated individually and then, RSM was used to find the optimized combination of additives for refolding of reteplase IB produced by E. coli (BL-21). Finally, the selected combination was used to compare the refolding efficiency of IBs obtained from three different E. coli strains (BL21 (DE3), Rosetta-gami, and SHuffel).
In addition, due to a previously published refolding procedure, the effect of freeze–thawing was investigated on reteplase refolding efficiency [19]. Our results indicated that freeze-thawing method has an adverse effect on the solubility of IBs and refolding efficiency.
Section snippets
Protein expression
For recombinant protein expression, E. coli strains BL21 (DE3), Rosetta-gami (DE3), and SHuffel T7 competent cells were transformed using standard protocol. As described in our previously published report, a codon optimized reteplase gene -cloned in pET-21a- was used during these experiments. Briefly, 5 ml of LB medium containing ampicillin was inoculated using a single colony of E. coli BL21 and incubated for 12 h at 37 °C, 180 rpm shaking. 200 μl of the pre-cultures were used for inoculation
Expression and refolding of reteplase in E. coli BL21 (DE3)
E. coli BL21 harboring the pET-ret plasmid was used for expression of reteplase. A remarkable amount of the recombinant protein was expressed after induction with 1 mM IPTG at 37 ֯C and 180 rpm shaking during 4 h. As shown in Fig. 2, reteplase was expressed and accumulated in the insoluble fraction. IBs were isolated and solubilized in 8 M urea. The refolding procedure was carried out using dilution strategy by gradual addition of solubilized IBs to refolding buffer in small droplets. In order
Discussion
In many cases, high-level expression of recombinant proteins in E. coli results in accumulation of proteins as aggregated IBs. The same happens for proteins such as reteplase with several disulfide bonds. Refolding is an approach to recover functionally active products from inactive structure of IBs. The main issue in refolding process is optimizing renaturation condition by preventing the formation of intermediate aggregates. There are numerous chemical additives that have been proved useful
Conclusion
In the present work we used RSM to find the optimum condition for in vitro refolding of reteplase from inclusion bodies produced by E. coli cells. The highest amount of active reteplase was recovered after dilution in buffer containing 1 mM GSH, 0.1 mM GSSG, 1 mM NH4OAc, 0.13 M histidine, 1.64 M methionine, 0.33 M cysteine and 0.34 M arginine. We also found that using genetically-modified E. coli strains cannot be helpful to increase the yield of reteplase refolding.
Author statement
Mehrnoosh Fathi-Roudsari: Conceptualization, Work Design, Laboratory supervision, Writing original manuscript, Software.
Amirhossein Maghsoudi: Scientific Consultant, Reviewing.
Nader Maghsoudi: Conceptualization, Scientific Consultant, Reviewing.
Sepideh Niazi: Laboratory assistance.
Morvarid Soleiman: Laboratory assistance, Software.
References (21)
- et al.
Purification and efficient refolding process for recombinant tissue-type plasminogen activator derivative (reteplase) using glycerol and Tranexamic acid
Process Biochem.
(2017) - et al.
Optimizing of the formation of active BMW-amylase after in vitro refolding
Protein Expres. Purif.
(2012) - et al.
Efficient refolding of recombinant lipase from Escherichia coli inclusion bodies by response surface methodology
Protein Expr. Purif.
(2010) - et al.
Modeling and optimizing of physicochemical features of walnut-oil beverage emulsions by implementation of response surface methodology: effect of preparation conditions on emulsion stability
Food Chem.
(2015) - et al.
Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review
Protein Expr. Purif.
(2014) - et al.
A novel protein refolding system using lauroyl-l-glutamate as a solubilizing detergent and arginine as a folding assisting agent
Protein Expr. Purif.
(2011) - et al.
Urea impedes the hydrophobic collapse of partially unfolded proteins
Biophys. J.
(2009) - et al.
Optimizing refolding condition for recombinant tissue plasminogen activator, Iran
J. Biotechnol.
(2011) - et al.
Studies on the refolding process of recombinant horseradish peroxidase
Mol. Biotechnol.
(2013) - et al.
Protein refolding using chemical refolding additives
Biotechnol. J.
(2013)
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2021, Nongye Gongcheng Xuebao/Transactions of the Chinese Society of Agricultural Engineering