Non-chromatographic purification of thermostable endoglucanase from Thermotoga maritima by fusion with a hydrophobic elastin-like polypeptide

Highlights

• Construct a small gene library of ELP from ELP₅ to ELP₅₀.

• Purify and produce the fusion EG12B-ELP₅₀ by inverse transition cycling.

• Improve the thermostability of EG12B by fusion with ELP₅₀.

• Provide a rapid and simple strategy for target protein purification.

Abstract

Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP₅ to ELP₅₀ was constructed using recursive directional ligation by plasmid reconstruction. ELP₅₀ was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP₅₀, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP₅₀ expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP₅₀ was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP₅₀ purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP₅₀ had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP₅₀ could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.

Introduction

Cellulose is the most abundant organic biopolymer on Earth, making it a significant renewable carbon resource [1]. The biodegradation of cellulose using cellulases is a promising and sustainable approach to obtain monomeric glucose subunits. Cellulase is the 3rd highest used enzyme in industry after proteases and amylases, and have proven applications in biofuel production, bio-deinking, textile industry, and food and feed processing industry [2]. Cellulases are classified into endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.9), and 1,4-β-glucosidases (EC 3.2.1.21) [3]. Endoglucanases can break the internal bonds of cellulose and disrupt its crystalline structure, exposing individual cellulose polysaccharide chains. Given the importance of cellulose biodegradation, the isolation and purification of endoglucanases have attracted much attention [4]. Endoglucanase EG12B from Thermotoga maritima is a thermophilic enzyme that could be suitable for industrial use [5]. Previously, EG12B has been expressed in engineered Escherichia coli and purified using column chromatography [6]. However, traditional protein purification using column chromatography has some disadvantages: it is time-consuming and expensive and has a limited capacity. Therefore, the need to develop a simple, inexpensive, and reliable purification procedure to produce high-purity EG12B at a large scale is urgently needed.

Expression of a fusion with another protein or peptide tag in E. coli is a widely used strategy for obtaining a target protein with high purity [7]. Elastin-like polypeptides (ELPs) are artificial biopolymers composed of short repeating peptide motifs. The most commonly used ELP sequence is the pentapeptide (VPGXG)_n, in which X is a guest residue, which can be any amino acid except proline [8]. ELPs exhibit stimulus-responsive reversible phase transition behavior and are soluble at temperatures below the inverse transition temperature (T_t). In contrast, ELPs aggregate into micron-sized coacervates above the T_t [9,10]. The T_t values of ELPs are dependent on the composition of the pentameric repeats as well as the number of repetitive units which are intrinsic parameters. Extrinsic parameters, such as the ELP concentration, pH, and salt effects also have a significant effect on the T_t [11].

ELPs can serve as simple protein tags to impart reversible phase transition behavior to the target ELP-fusion protein, providing an inexpensive batch method for the purification of recombinant proteins by inverse transition cycling (ITC) [12,13]. Crucially, this process does not require the use of chromatographic methods. If the free protein is required, a protease recognition site can be inserted between the protein and ELPs, and the ELP tag can be subsequently removed through an additional round of ITC after protease digestion [14].

Here, a new strategy for the production of high-purity EG12B was developed. We chose hydrophobic valine as a guest residue and constructed a small ELP gene library using recursive directional ligation by plasmid reconstruction. ELP₅₀, which has an appropriate molecular weight and T_t, was fused at the C-terminus of EG12B to simplify the isolation and purification of the target protein using a non-chromatographic ITC method.