Team:ShanghaiTechChina B/Project/EGF
EGF
Over the past few months, our team worked primarily with E.colis, which are ranked in the organisms of Level 1 risk group. We attempted to utilize our engineered bacteria to help treat IBD, so we considered as much as we can to ensure the safety of our entire project--not only the design of our therapeutic methods, but also our lab work.
Introduction
Human epidermal growth factor (hEGF) is a 6045-Da protein with 53 amino acid residues. It can stimulate cell growth, proliferation and differentiation by binding to its receptor EGFR both in vivo and vitro. Some people utilize it as cosmetic product because of the potent ability of recovering epithelium.
New treatment of IBD—EGF enema
The study by Atul Sinha etc. in 2003 suggested that EGF enema is an effective treatment for active left-sided ulcerative colitis (a kind of Inflammatory Bowel Disease) . In their experiments, 12 patients with left-sided ulcerative colitis received daily enemas of 5 μg of recombinant hEGF (expressed in Saccharomyces cerevisiae) in 100 ml of an inert carrier and 12 received daily enemas with carrier alone. Patients were assessed clinically by sigmoidoscopy and biopsy. After 2 weeks, 83% of patients given EGF enemas were in remission, as compared with 8% in the control group.
EGF expression
We were inspired by the above research and designed a bacterial strain that can secrete EGF in patients’ gut. E.coli. has been chosen as expression strain because it is aborigines in human’s gut and easy to be programing. A signal peptide phoA is added at the N-terminus to facilitate secretion. We engineered two peptide tags, a phoA signal peptide at the N-terminus and a FLAG tag at the C-terminus of hEGF protein. PhoA is a fragment of signal peptide that guides the protein to go across cytomembrane and do not decrease activity of the protein. FLAG-tag is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK. The ID of this part is BBa_K2152001
Physiological activity of recombinant EGF
In order to test the physiological activity of recombinant EGF, we substituted FLAG tag for 6*HIS tag (part ID: BBa_K2152000) and purified it by Ni-NTA Agarose. The purified recombinant protein was applied to mouse models of intestinal inflammation by enemas (performing by SYSU-MEDICINE, click here to get more information. The result shows that recombinant EGF enemas are an effective treatment for intestinal inflammation in mouse.
