High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.
Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.
Su
Lin
L
National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
Chen
Song-Sen
SS
Yang
Ke-Gong
KG
Liu
Chang-Zheng
CZ
Zhang
Yan-Li
YL
Liang
Zhi-Quan
ZQ
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003114:Colony-Forming Units Assay; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000494:pharmacology; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D004921:Erythropoietin