A combination strategy of solubility enhancers for effective production of soluble and bioactive human enterokinase

Highlights

• Solubility enhancers are incorporated into the N-terminus of enterokinases for promoting soluble expression in bacteria.

• Typically engineered enterokinases show low expression level or partially decreased enzymatic activity (−35%).

• Tandem combinations of fusion proteins can facilitate effective production of functional enterokinase (~ 100 mg/L culture).

• The resulting enterokinases exhibit significantly enhanced solubility (above 90%) and enzymatic activity.

Abstract

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

Introduction

Along with optimization of biological activity, soluble expression of recombinant active proteins is one of the major issues to be considered in the field of biotechnology, in terms of cost-effectiveness and mass production of biological reagents. However, some valuable proteins, mostly derived from human and other eukaryotes, are prone to form insoluble and inactive aggregates while using a bacterial expression system due to improper protein folding and modification (Butt et al., 2005, Pacheco et al., 2012). To address this challenge, several advanced strategies have been introduced to achieve effective and stable expression of soluble recombinant proteins (Khow and Suntrarachun, 2012). Among them, the use of solubilizing fusion partners including maltose-binding proteins (MBP) (Dyson et al., 2004, Kapust and Waugh, 1999, Song et al., 2012) and glutathione S-transferase (GST) (Rabhi-Essafi et al., 2007) has been recognized as the primary option for improving protein solubility and expression level. In addition, fusion tags can be utilized for facile purification of expressed proteins by affinity chromatography, allowing high-yield purification with remarkable purity (Waugh, 2005). Despite these advantages, the removal of the fusion partners from purified proteins is highly recommended for further applications because they can inhibit the biological function of recombinant proteins and trigger unwanted immune responses when used as a therapeutic agent (Choi et al., 2001). For this process, a variety of endopeptidases have been commonly used for cleavage of pre-designed peptide linkers located between the solubilizing protein and the protein-of-interest (Waugh, 2011).

Hydrolases are a group of hydrolytic enzymes that promote the breakdown of various biochemical bonds. Among them, protease (peptidase) has a specialized role in cleavage of amide bonds in peptides and proteins to precisely regulate biological processes and to maintain homeostasis (Mahesh et al., 2018). Enterokinase, also known as enteropeptidase, belongs to a class of serine proteases and is a disulfide-linked heterodimeric enzyme, composed of a heavy chain (87 kDa) and a light chain (26 kDa) (Kitamoto et al., 1994). In the small intestine, enterokinase is mainly produced in the duodenal brush borders for activation of the digestive system by converting proenzyme trypsinogen into active proteolytic enzyme, trypsin (Lobley et al., 1973, Yamashina, 1956). Interestingly, the light chain of enterokinase exhibits a remarkable target specificity and catalytic activity, enabling the effective recognition of a pentapeptide sequence Asp-Asp-Asp-Asp-Lys (DDDDK) and hydrolysis of the bond located after the lysine residue (Light and Janska, 1989). With this site-specific activity, enterokinase is found to be stable over a broad range of temperature (4–45 °C) and pH (4.5–9.5) (Gasparian et al., 2006). Based on these properties, enterokinase has been widely applied as a key enzyme for the process of isolating and removing the solubilizing fusion partners or affinity tags from purified recombinant proteins (Choi et al., 2001). However, several difficulties exist in enterokinase production. Previous studies reported that overexpressed enterokinase in bacteria was dominantly expressed in insoluble aggregates and formed inclusion bodies, resulting in extremely low production yields of soluble enterokinase (Gasparian et al., 2006, Niu et al., 2015). In order to address this limitation, a refolding method was introduced and implemented despite the apparent disadvantage of a complicated and inconvenient process (Yi and Zhang, 2006, Tan et al., 2007). In addition, periplasmic expression of enterokinase was attempted but showed low expression levels (Collins-Racie et al., 1995). Due to the difficulty of obtaining recombinant enterokinase in bacteria, most of the commercial enterokinases are expressed in yeast or extracted from the intestine of animals with high production costs (Melicherová et al., 2017).

This study presents a soluble expression of recombinant enterokinase light chain (EKL) in Escherichia coli (E. coli) by using several solubility-enhancing tags (Scheme 1). As human enterokinase is known to have 10 times higher catalytic activity than animal enterokinase, human enterokinase is selected for this study and further applications (Gasparian et al., 2006). As shown in Fig. S1, unmodified enterokinases exhibit poor solubility and low expression levels in E. coli. To increase solubility of expressed enterokinases, we employed two types of fusion proteins including maltose binding protein (MBP) (di Guan et al., 1988) and phosphoglycerate kinase (PGK) (Park et al., 2008, Song et al., 2012). Thioredoxin (Trx) was also used to facilitate correct disulfide-bond formation and protein folding, as enterokinase contains eight cysteine residues (Nordström, 1972). These protein tags were genetically incorporated into the N-terminus of enterokinases in various combinations. The resulting enterokinases were highly expressed in E. coli as a soluble form with proteolytic activity comparable to a commercial protein, as the details demonstrated herein.