title: Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli.
abstract: The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98%) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.
authors.affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury of PLA, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, People's Republic of China.
authors.forename: Song
authors.initials: S
authors.lastname: Wang
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Shen
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.forename: Mo
authors.initials: M
authors.lastname: Chen
authors.forename: Shilei
authors.initials: S
authors.lastname: Chen
authors.forename: Aiping
authors.initials: A
authors.lastname: Wang
authors.forename: Zhou
authors.initials: Z
authors.lastname: Zhang
authors.forename: Xinze
authors.initials: X
authors.lastname: Ran
authors.forename: Tianmin
authors.initials: T
authors.lastname: Cheng
authors.forename: Yongping
authors.initials: Y
authors.lastname: Su
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D019382:Human Growth Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D055016:Megakaryocyte Progenitor Cells / Q000187:drug effects; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; C515728:thrombopoietin mimetic peptide; D019382:Human Growth Hormone