title: High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.
abstract: 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.
authors.affiliation: 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.
authors.forename: Lin
authors.initials: L
authors.lastname: Su
authors.forename: Song-Sen
authors.initials: SS
authors.lastname: Chen
authors.forename: Ke-Gong
authors.initials: KG
authors.lastname: Yang
authors.forename: Chang-Zheng
authors.initials: CZ
authors.lastname: Liu
authors.forename: Yan-Li
authors.initials: YL
authors.lastname: Zhang
authors.forename: Zhi-Quan
authors.initials: ZQ
authors.lastname: Liang
mesh_terms: 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
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D004921:Erythropoietin