Journal Article

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

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

2021

Overview

Background Proteins 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 . Results A 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. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli .

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Affinity chromatography / Alcohol Dehydrogenase - biosynthesis / Alcohol Dehydrogenase - isolation & purification / Analysis / Applied Microbiology / Bacterial Proteins - biosynthesis / Bacterial Proteins - isolation & purification / Biological activity / Biotechnology / Bone Morphogenetic Protein 7 - biosynthesis / Bone Morphogenetic Protein 7 - isolation & purification / Carbohydrate-binding module / Carbohydrates / Carbohydrates - chemistry / Carrier Proteins - biosynthesis / Carrier Proteins - isolation & purification / Cellulose / Chemistry / Chemistry and Materials Science / Cloning, Molecular / Control / E coli / Enzymes / Enzymology / Epidermal growth factor / Epidermal Growth Factor - biosynthesis / Epidermal Growth Factor - isolation & purification / Escherichia coli / Escherichia coli - metabolism / Fungal Proteins - biosynthesis / Fungal Proteins - isolation & purification / Fusion protein / Fusion tag / Gene Expression / Genetic Engineering / Glutathione / Glutathione transferase / Humans / Hydrolase / Hydrolases - biosynthesis / Hydrolases - isolation & purification / Identification and classification / Inclusion bodies / Inclusion Bodies - metabolism / Levan / Levan-agarose / Lipase - biosynthesis / Lipase - isolation & purification / Maltose / Maltose-binding protein / Maltose-Binding Proteins / Methods / Microbial Genetics and Genomics / Microbiology / Microorganisms / Modules / Peptides / Polyethylene terephthalate / Polypeptides / Protein engineering / Protein Engineering - methods / Protein expression / Protein folding / Protein Processing, Post-Translational / Protein purification / Proteins / Recombinant Fusion Proteins - biosynthesis / Recombinant Fusion Proteins - chemistry / Recombinant Fusion Proteins - genetics / Recombinant Fusion Proteins - isolation & purification / Solubility / Solubilization / Soluble expression / Tags / Vascular endothelial growth factor / Vascular Endothelial Growth Factor A - biosynthesis / Vascular Endothelial Growth Factor A - isolation & purification