Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

Anh Ngoc Nguyen,Yeon Jin Jang,Han Choe,Jung-A Song,Mark J Osborn,Grace G Kwon,Heung-Bum Oh,Thu Trang Thi Vu,Sang Su Park,Minh Tan Nguyen,Jonghwa Jin,Bich Hang Do,Jiwon Yoo,Jaepyeong Jang

doi:10.1038/s41598-017-16167-x

Abstract

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a β-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

Highlights

Human fibroblast growth factor 21 has been characterized as an important regulator of glucose and lipid metabolism homeostasis
The Human fibroblast growth factor 21 (hFGF21) fusion vectors were transformed into the E. coli BL21(DE3) to characterise the behaviour of the fusion proteins expressed in the cytoplasm
At 37°C, only the maltose-binding protein (MBP) and protein disulphide isomerase (PDI) tags could enhance the solubility to greater than 60%, whereas the other fusion proteins were primarily expressed as inclusion bodies

Summary

Introduction

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. To produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). Eight tag genes were utilised: hexahistidine (His6), small ubiquitin-related modifier (Sumo), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). To obtain bioactive hFGF21, the MBP-hFGF21 fusion was purified by a single conventional chromatography technique followed by tag cleavage This method resulted in a maximal yield of 8.1 mg of hFGF21 from 500 mL of cell culture in highly pure form with negligible endotoxin levels. The FGF21 purified under these novel conditions increased the proliferation of NIH-3T3 cells transfected with β-klotho proving functionality of the recombinant protein

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