Journal: Infection and Immunity
Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿
Figure Lengend Snippet: Construction and expression analysis of Pvs25H in Pichia pastoris . (a) The 5′- and 3′-coding regions of the Pvs25 gene sequence with its predicted amino acid sequence (Ala 23 to Leu 195 ). The Pvs25 gene fused to a hexahistidine tag at its C terminus was inserted into the SnaBI and EcoRI sites of pPIC9K, and the gene was integrated into the chromosomal DNA of P. pastoris strain GS115 by homologous recombination. (b) After selection of a high-producer clone that secreted the recombinant protein, nickel-affinity chromatography-purified Pvs25H was analyzed by SDS-PAGE (lane 1). M, molecular marker. (c) The affinity-purified Pvs25H was fractionated by size exclusion chromatography from which at least five chromatographic peaks were observed. (d) Size exclusion chromatography fractions 14-26 were subjected to SDS-PAGE (15% acrylamide). Based on the apparent molecular mass of each protein band, the fractions 16, 17, 18-19, 21-22, and 22-23 were defined as the tetramer, trimer, dimer, monomer B, and monomer A, respectively. (e) Selected chromatography peaks were subjected to 5 to 20% acrylamide gradient SDS-PAGE under nonreducing or reducing (10% 2-ME and boiling) conditions. (f) Hydrophobic interaction chromatography of the Pvs25H-A and Pvs25H-B monomeric isoforms. Elution fractions (A) are eluates of 2 M NH 4 SO 4 (Pvs25H-A) and elution fractions (B) are eluates of PBS (Pvs25H-B). (g) Each isoform (M, a mixture of dimer, trimer and tetramer; B, B isoform; A, A isoform) was subjected to 15% acrylamide SDS-PAGE under various conditions, as indicated. (h) The N-terminal protein sequence of each isoform. Positively numbered amino acid residues (Ala 1 and the following) are residues of the Pvs25 protein; negatively numbered amino acids are derivatives of the pPIC9K α-factor secretion signal. The A monomer revealed a single, unique sequence, but the B monomer and the multimers showed a mixture of different N termini.
Article Snippet: The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ).
Techniques: Expressing, Sequencing, Homologous Recombination, Selection, Recombinant, Affinity Chromatography, Purification, SDS Page, Marker, Affinity Purification, Size-exclusion Chromatography, Chromatography, Hydrophobic Interaction Chromatography