title: Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli.
abstract: Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, PR China.
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Zang
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.forename: Dawen
authors.initials: D
authors.lastname: Yuan
authors.forename: Yumin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.forename: Haiqin
authors.initials: H
authors.lastname: Lu
authors.forename: Chao
authors.initials: C
authors.lastname: Chang
authors.forename: Junchuan
authors.initials: J
authors.lastname: Qin
mesh_terms: D002460:Cell Line; D018835:Chaperonin 10 / Q000096:biosynthesis; D018834:Chaperonin 60 / Q000096:biosynthesis; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D006801:Humans; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D034061:Thrombopoiesis / Q000187:drug effects / Q000502:physiology; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase