[Expression of TPO mimetic peptide chimeric proteins with human IgG1 Fc fragments and their biological characters].
Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids. By transforming the four recombinant plasmids into E. coli. BL21 (DE3) respectively, we got 3 kinds of engineered E. coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column. The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering.
Li
Yue-Xi
YX
East China Institute of Medical Biotechniques, Nanjing 210002, China. liyxi@163.net
Li
Chao
C
Tao
Kai-Hua
KH
Jia
Xiang-Hong
XH
Cheng
Du-Sheng
DS
Huang
Pei-Tang
PT
D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D005260:Female; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D013921:Thrombocytopenia / Q000097:blood / Q000276:immunology; D013926:Thrombopoietin / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D013926:Thrombopoietin