Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis.
In the present study, the mature epidermal growth factor (EGF) protein was engineered to incorporate a high affinity collagen-binding domain (CBD) derived from co-agulation von Willebrand factor, to specifically target EGF to colonic lesions. The fusion protein was expressed in an E. coli bacterial expression system, purified by metal chelate chromatography, and renatured by oxidative refolding into a soluble biologically active growth factor. The EGF-CBD fusion protein bound tightly to collagen matrices under conditions in which native non-targeted EGF was washed away. In biologic assays, the EGF-CBD fusion protein stimulated NIH3T3 cell proliferation with near wild-type biological activity. In vivo binding studies showed that the collagen-targeted EGF, but not the non-targeted EGF, accumulated at areas of exposed collagen on the luminal surface of the inflamed colon. Finally, a single colonic instillation of the collagen-targeted EGF-induced a more rapid regeneration of intestinal crypts 24 h after treatment (no. of crypts = 89.2+/-8.1) compared to the non-targeted EGF (no. of crypts = 52.2+/-29.8; p=0.027), and the PBS control (no. of crypts = 24. 0+/-22.9; p=0.001). Taken together, these findings indicate that intracolonic delivery of collagen-targeted EGF represents a potentially effective therapeutic strategy for acute or chronic inflammatory bowel disease.
Hall
F L
FL
Division of Colon and Rectal Surgery, Keck School of Medicine of USC, Los Angeles, CA 90089, USA.
Kaiser
A
A
Liu
L
L
Chen
Z H
ZH
Hu
J
J
Nimni
M E
ME
Beart
R W
RW
Gordon
E M
EM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D003093:Colitis, Ulcerative / Q000188:drug therapy* / Q000473:pathology; D003094:Collagen / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000473:pathology; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004815:Epidermal Growth Factor / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression; D007413:Intestinal Mucosa / Q000187:drug effects / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012107:Research Design; D014841:von Willebrand Factor / Q000235:genetics / Q000494:pharmacology*
D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D004815:Epidermal Growth Factor; D003094:Collagen