Overexpression of hTFF2 in the pET system and its in vitro pharmacological characterization.
The trefoil factor family 2 (TFF2), a member of the trefoil factor family, plays a critical role in the defense and repair of gastrointestinal mucosa. However, its widespread application is hampered by difficulties in large-scale production of the recombinant protein suitable for clinical use. The aim of the present study was to produce hTFF2 by Escherichia coli expression system and explore its in vitro pharmacological characterization. hTFF2 gene encoding mature peptide was obtained by RT-PCR, and then inserted into the expression vector pET32a to construct the recombinant pET32a-hTFF2. After confirmation by gene sequencing, pET32a-hTFF2 was transformed into E. coli Origami B(DE3), and TrxA-hTFF2 fusion protein was expressed by conventional IPTG induction in a shake flask and analyzed with SDS-PAGE and Western-blot. Subsequently, TrxA-hTFF2 was isolated by Ni-NTA affinity chromatography, and ultrafiltration. Finally, we tested the effect of hTFF2 on cell migration in an in vitro restitution model and cell proliferation by MTT assay. The data revealed that the recombinant vector pET32a-hTFF2 was constructed successfully. TrxA-hTFF2 fusion protein was expressed to 246.5mg/L and its purity was above 95% after purification. SDS-PAGE and Western blot analyses showed that the fusion protein presented as a single band with a molecular weight of 32kDa. In vitro model of wounding demonstrated that hTFF2 enhanced migration activity by three folds. MTT assay exhibited a statistically significant dose-dependent growth-enhanced effect. Collectively, the results suggest that the recombinant hTFF2 was expressed in E. coli with high production, purity and biological activity.
Sun
Yong
Y
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, The Third Military Medical University, Gaotanyan Street, Chongqing 400038, PR China.
Wu
Wei
W
Wang
Lin
L
Liang
Guangping
G
Zhang
Yong
Y
Lv
Shangjun
S
Wang
Zhen
Z
Wang
Shiliang
S
Peng
Xi
X
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005753:Gastric Mucosa / Q000187:drug effects* / Q000473:pathology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010455:Peptides / Q000008:administration & dosage / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071164:Trefoil Factor-2; D014945:Wound Healing / Q000187:drug effects*
D010455:Peptides; D011993:Recombinant Fusion Proteins; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2