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

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.

Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

“…It is especially important when a mammalian protein of interest is produced in a heterologous, such as bacterial, host. GFs are a protein type produced in a heterologous host [1,[15][16][17][18][19][20]. To meet increasing demand, highly productive systems, such as E. coli or yeast, are used for GF production.…”

“…Growth factors (GFs) are signaling proteins that positively regulate various cellular processes by binding appropriate receptors on the cell surface. The signaling pathways induced by GFs regulate cellular responses, such as growth, differentiation, proliferation, maintenance, in ammation, and angiogenesis [1,2]. Traditionally, epidermal GF (EGF), vascular endothelial GF (VEGF), keratinocyte GF (KGF), insulin-like GF (IGF), and broblast GF (FGF) family members are used for wound healing, curing and targeting diseases, and stimulating hair growth [3][4][5][6][7][8][9][10].…”

“…With an increasing interest in functional cosmetics, the demand for GFs has also increased. Various expression systems, including Escherichia coli [1,[15][16][17][18][19], Bacillus subtilis [20], mammalian cell [21], baculovirus [22], silkworm [23,24], and plant [11,12] systems, are used for cost-effective and e cient production of recombinant GFs. Among them, the E. coli system is frequently used for the production of recombinant proteins because of facile genetic modi cation, rapid protein expression, and high growth rate.…”

“…To overcome the mispairing of disul de bonds, E. coli host strains have been genetically engineered to control the cellular redox environment [26][27][28]. As another strategy, solubility enhancers, such as maltose-binding protein (MBP) [1,17], oleosin [12], low-molecular-weight protamine [14], HaloTag [18], glutathione S-transferase [1,17], protein disul de isomerase (PDI) [1,17], ELK16 [29], and thioredoxin [1,17,30], are fused to the target protein, including GFs. As the solubility of the tag affects the solubility of the passenger protein, choosing an appropriate solubility tag is critical [31].…”

“…As the solubility of the tag affects the solubility of the passenger protein, choosing an appropriate solubility tag is critical [31]. Nguyen et al [1,17] reported that seven protein tags, namely, thioredoxin, glutathione S-transferase, N-utilization substance protein A, the b'a'domain of PDI (PDIb'a'), PDI, 6⋅His, and MBP, were tested, and some or all of the protein tags enhance the solubility of the fusion protein in E. coli.…”