A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Hyunjun Ko,Mi-Jin Kim,Jung-Hoon Bae,Jiyeon Yi,Bong Hyun Sung,Jin Kang,Jung-Hoon Sohn,Minsik Kang

doi:10.1186/s12934-021-01725-w

Abstract

BackgroundProteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli.ResultsA novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused.ConclusionsThe use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Highlights

Proteins with novel functions or advanced activities developed by various protein engineering tech‐ niques must have sufficient solubility to retain their bioactivity
Construction of a plasmid for the expression of carbohydrate-binding module 66 (CBM66)‐fused proteins To express target proteins fused with the CBM66 tag, the plasmid pCBM66 was constructed using the pET21b vector backbone
The target protein was designed to be expressed with a CBM66 tag on the N-terminus under the T7 promoter

Summary

Introduction

Proteins with novel functions or advanced activities developed by various protein engineering tech‐ niques must have sufficient solubility to retain their bioactivity. Escherichia coli is a predominant workhorse in a wide range of biotechnological applications It has been employed as an efficient cell factory for the production of biomolecules, including high-value recombinant proteins. The inclusion body should be solubilized and refolded by the strong and large amount of detergents to recover the bioactivity of the target protein, which is a hurdle for scale-up production [1,2,3]. The latter comprises several strategies, including the optimization of culture conditions, host genome engineering, and the application of fusion tags to increase the solubility of target proteins [4]

Methods

Results

Conclusion