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

Abstract

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.

Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification

“…Another main concern with the large-scale preparation of industrial enzymes is the yield of the target protein. Previously, Is PETase was expressed in E. coil with a novel fusion tag, carbohydrate-binding module 66 (CBM66) to improve the soluble expression, and the titer of the recombinant Is PETase reached 370 mg/L 28 . Is PETase was also expressed with microalgaes as host, such as P. tricornutum and C. reinhardtii and the recombinant protein can be detected with western blot 29 .…”

Efficient virus detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis

“…One such example are carbohydrate-active proteins like CBH1 from Trichoderma reesei ( Zhong et al 2011 ). As a positive side-effect, these protein tags can even enhance the solubility like shown for carbohydrate-binding module 66 (CBM66) in E. coli ( Ko et al 2021 ). In our study, different combinations of nanobody and Cts1 carrier resulted in varying levels of expression, secretion and activity.…”

Advances in biosynthesis of peptide drugs: Technology and industrialization

“…Consequently, to expand the expression capacity of peptide drugs in E. coli, new fusion tags need to be investigated. Ko et al [55] developed a novel carbohydrate-binding module 66 (CBM66) fusion tag to increase the soluble expression of heterologous proteins in E. coli. Compared to MBP and GST tags, the solubilizing effect of CBM66 tag on poly(ethylene terephthalate) hydrolase (PETase) showed a 3.7 times increase.…”