title: A biologically active vMIP-II-IgG3-TfN fusion protein, secreted from methylotrophic yeast Pichia pastoris.
abstract: The viral macrophage inflammatory protein II (vMIP-II) which showed a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus. To explore the feasibility of using TfN as a carrier moiety for delivery of therapeutic proteins, a genetically engineered vMIP-II-IgG3-TfN fusion gene was loaded into the yeast expression vector pPICZα. The linearized recombinant plasmid pPICZα-vMIP-II-IgG3-TfN was transformed into X33 competent cells. The recombinant protein was expressed in methylotrophic yeast Pichia pastoris and was confirmed to have expected molecular mass of 48 kDa by SDS-PAGE. Using methods combining ammonium sulfate precipitation, dialysis, ultrafiltration and affinity chromatography, the vMIP-II-IgG3-TfN fusion protein was successfully purified from the supernatant of the broth. Western-blotting analysis showed that 6× His antibody recognized the purified vMIP-II-IgG3-TfN. CD spectrum revealed a positive peak at 196.5 nm and a negative peak at 209 nm. MALDI-TOF MS analysis showed that the purified vMIP-II-IgG3-TfN was an intact and homogeneous protein. The pepsin digestibility assay showed that the vMIP-II-IgG3-TfN fusion protein could be digested into small fragments by pepsin after 2 min treatment. The vMIP-II-IgG3-TfN fusion protein was found to be stable in human plasma for up to 48 h. Furthermore, in vitro bioactivity assay indicated that the vMIP-II-IgG3-TfN fusion protein can block the chemotaxis of U937 cells induced by SDF1α. In total, this study illustrates the development of an active vMIP-II-IgG3-TfN fusion protein expressed in P. pastoris.
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Xiu-ying
authors.initials: XY
authors.lastname: Li
authors.forename: Xue-mei
authors.initials: XM
authors.lastname: Mo
authors.forename: Guang
authors.initials: G
authors.lastname: Zhang
authors.forename: Han-xiao
authors.initials: HX
authors.lastname: Sun
mesh_terms: D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000037:antagonists & inhibitors / Q000502:physiology; D018925:Chemokines / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005590:Fractional Precipitation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D010434:Pepsin A / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010843:Pichia; D055550:Protein Stability; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D014168:Transferrin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D054377:Chemokine CXCL12; D018925:Chemokines; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014168:Transferrin; C108059:vMIP-II; D010434:Pepsin A