Human growth hormone-transferrin fusion protein for oral delivery in hypophysectomized rats.
Transferrin (Tf)-based recombinant fusion protein approach was investigated to achieve oral delivery for human growth hormone (hGH). Plasmid constructs expressing the fusion proteins were established by fusing coding sequences of both hGH and Tf in frame. Fusion proteins were produced in serum free media by transient transfection of human embryonic kidney HEK293 cells. The SDS-PAGE analysis of conditioned media showed that fusion proteins expressed at high purity with a 100 kDa molecular weight; the Western blot analysis with anti-hGH and anti-Tf antibodies verified the identity of fusion proteins. The Nb2 cell proliferation and Caco-2 cell Tf receptor (TfR) binding assays demonstrated that fusion proteins retained bioactivity of both hGH and Tf, respectively. A helical linker was inserted as spacer between hGH- and Tf-domain to enhance the bioactivity and the yield of the fusion protein. Two fusion proteins, hGH-Tf (GT) and hGH-(H4)(2)-Tf (GHT) were obtained and assessed in hGH-deficient hypophysectomized rats for in vivo biological activity. Results from seven-day subcutaneous dosing (1.25mg/kg/day) demonstrated that both GT and GHT fusion proteins were bioactive in vivo, comparable to native hGH. However, only the GHT, but not GT, fusion protein promoted a modest but statistically significant weight gain after oral dosing with 12.5mg/kg/day.
Amet
Nurmamet
N
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA.
Wang
Wei
W
Shen
Wei-Chiang
WC
D000284:Administration, Oral; D000818:Animals; D015153:Blotting, Western; D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005240:Feasibility Studies; D005260:Female; D020249:Hormone Replacement Therapy; D019382:Human Growth Hormone / Q000008:administration & dosage* / Q000737:chemistry; D006801:Humans; D007016:Hypophysectomy; D007279:Injections, Subcutaneous; D023421:Models, Animal; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011990:Receptors, Transferrin / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D015430:Weight Gain / Q000187:drug effects
D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D019382:Human Growth Hormone