Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

Figure 1 Cloning of hdHGF cDNA and construction of expression vectors pUC-hdHGF and pPIC9-hdHGF.

Figure 2 Schematic representation of hdHGF expression cassettes used. Arrowhead indicates the cleavage site of Kex2 protease.

Figure 3 Analysis of PCR products by 1% agarose electrophoresis (A and B). Lane 1: fragment (F2) amplified with P3-P4 primer pair; lane 2: fragment (F1) amplified with P1-P2 primer pair; lane 3: fragment (whole length) amplified with M1-M2 primer pair; M1: 200-bp ladder marker; M2: λ+HindIII marker. The arrowheads show the bands of the interesting fragments

Figure 4 Agarose electrophoresis analysis of restriction enzyme mapping of recombinant plasmids of pPIC9-hdHGF. Lanes 1-5: recombinant plasmids of pPIC9-hdHGF digested by BamHI and NotI; lane 6: λ-DNA/HindIII marker. The arrowhead reveals bands of the interesting fragment.

Figure 5 Fifteen percentage SDS-PAGE analysis of high expression strains in shake-flask culture. Lane 1: LMW marker; lane 2: GH209; lane 3: HG211; lane 4: HG305; lane 5:HG309; lane 6: GS115. The arrowhead shows bands of the interesting protein.

Figure 6 Fifteen percentage SDS-PAGE and Western blotting analysis of the 5-L fed-batch high cell density fermentation of GH209. Lane 1: LMW marker; lane 2: 10% SDS-PAGE result; lanes 3 and 4: Western blotting result. The arrowhead shows bands of the interesting protein.