Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.
Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.
Kuai
L
L
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Jiangsu, China.
Wu
C
C
Qiu
Q
Q
Zhang
J
J
Zhou
A
A
Wang
S
S
Zhang
H
H
Song
Q
Q
Liao
S
S
Han
Y
Y
Liu
J
J
Ma
Z
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry* / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*
D017931:DNA Primers; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1; D004921:Erythropoietin