High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

Abstract

Background: Fibroblast growth factor 21 (FGF21) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21) in Escherichia coli (E. coli) is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in E. coli BL21(DE3).

Results: By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection.

Conclusions: This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

Figure 1

Schematic illustration of SUMO-FGF21 synthesis. According to the human FGF21 mRNA coding sequence and the SUMO fragment C-terminal sequence, 21 primers were designed and synthesized. As shown in Figure 1, the SUMO-FGF21 was synthesized by primer extension and PCR. The detailed steps are reported in Material and Methods.

Figure 2

Synthesis of SUMO-FGF21 by PCR. The strategy for synthesizing the fusion fragment is described in Materials and Methods. The molecular weight of the PCR fragment containing SUMO-FGF21 is shown in Lane 1, 2 and 3. It is 864 bp in length.

Figure 3

SDS-PAGE analysis of expression and purification of SUMO-FGF21. Bacterial containing pET-SUMO-FGF21 were induced by IPTG for 3 h or 4 h at 37°C in Figure 3A. The results from the different treatments are shown here. M: protein molecular weight markers;Lane 1: the supernatant of BL21 (DE3) containing pET-SUMO-FGF21 without IPTG;Lane 2: the supernatant of BL21 (DE3) containing pET- SUMO-FGF21 induced with 1 mM IPTG for 3 h; Lane 3: the supernatant of BL21 (DE3) containing pET- SUMO-FGF21 induced with 1 mM IPTG for 4 h; Lane 4: the precipitation of BL21 (DE3) containing pET-SUMO-FGF21 induced with 1 mM IPTG for 4 h. After recombinant bacteria were treated by sonication and centrifugation, the supernatants were loaded on a DEAE sepharose FF and Ni-NTA orderly. The purification results for SUMO-FGF21 are shown in the Figure 3B. Lane1: protein molecular weight marker, Lane2: purified SUMO-FGF21 eluted from Ni-NTA column; Lane3: SUMO-FGF21 eluted from DEAE sepharose FF column.

Figure 4

SDS-PAGE analysis of SUMO-FGF21 by SUMO protease and rFGF21 assay by Western blot. The purified SUMO-FGF21 was digested by SUMO protease at 4°C overnight. As seen in Figure 4A, rFGF21 was cut from the SUMO-FGF21 by SUMO protease. Lane1: Protein Molecular Weight Marker. Lane2: purified rFGF21;Lane3: SUMO-FGF21 fusion protein after treatment with SUMO protease. Western blot analysis was performed as described in Materials and Methods. Immunoacitivity of purified rFGF21 with anti-human FGF21 antibody is shown in Figure 4B. Lane1: standard FGF21 (PeproTech Comp.);Lane2: purified rFGF21.

Figure 5

HPLC analysis of purified rFGF21. The purity of rFGF21 eluted from a Ni-NTA sepharose column was further evaluated by HPLC analysis using a C18 column. As seen from the chromatogram, the y-axis indicates the absorbance, while the x-axis represents elution time (min). The main peak observed at 14.984 min corresponds to rFGF21.

Figure 6

The effects of rFGF21 on reducing plasma glucose in diabetic rats. The STZ treated diabetic rats were injected with different doses of rFGF21 once daily for two weeks. Control: 0.9% NaCl; Low: 0.15 mg/kg rFGF21; Medium: 0.3 mg/kg rFGF21; High: 0.6 mg/kg rFGF21; Insulin: 5U per rat. The results show that rFGF21 acts in a dose-dependent manner to reduce plasma glucose in diabetic rats, with the high dose rFGF21 demonstrating better effects than the other doses. Compared to rFGF21, insulin displays a more significant effect in decreasing plasma glucose. ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001 were considered as the significant difference for post-treatment compared to pre-treatment.