Production of recombinant porcine colipase secreted by Pichia pastoris and its application to improve dietary fat digestion and growth of postweaning piglets

Abstract

Recombinant porcine pancreatic colipase (pCoL) was produced in the methylotrophic yeast Pichia pastoris. A synthetic yeast secretion cassette was constructed with the constitutive promoter of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene and the yeast a-mating factor signal peptide. The pCoL cDNA corresponding in the coding sequence, excluding a 16-amino acid segment of the native signal sequence, was cloned into the pGAPZaB vector and integrated into the genome of P. pastoris. Yeast transformants were cultured and bioactive pCoL protein was detected in the supernatant at a high-level of 126.8 mg/L after 3 days of culture. The transformed yeast containing the highest recombinant colipase level (pCoL yeast) and native yeast GS 115 not containing pCoL (non-pCoL yeast, as a control group) were separately cultured and the supernatants were adsorbed by dried skim milk. In an animal trial, two concentrations of colipase activity (0 vs. 5,000 U/kg in the diet) were blended with the pig corn-soybean basal diet and fed to weaned piglets for 4 weeks. The pCoL-administrated test group gained significantly more weight than piglets in the control group when measured at Day 15 (11.84 AE 0.70 vs. 10.59 AE 0.39 kg, P \ 0.05), Day 22 (15.84 AE 0.95 vs. 14.32 AE 0.59 kg, P \ 0.01), and Day 28 (20.19 AE 1.47 vs. 18.54 AE 0.92 kg, P \ 0.01) after weaning. The blood triglyceride (TG) concentrations were significantly increased in the experimental test group that received recombinant colipase on the 28th day of postweaning when compared with that of the control group (32.50 vs. 16.37 mg/dL; P \ 0.0001). These experimental data suggest that the use of recombinant porcine colipase as a dietary supplement provides an alternative approach for improving fat digestion and enhancing growth in postweaning piglets.

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“…To analyze cell proliferation, the LX-2 cells were seeded into 96-well culture plates at a cell density of 1 Â 10 4 cells/well for 24 h. The cells were then treated with several concentrations of GZT. The number of cells per well was estimated by flow cytometry using a FACS Calibur (Beccton Dickinson) after 24 or 72 h (Liu et al, 2008). A terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay was performed using the Fluorescein FragEL DNA Fragmentation Detection Kit according to the manufacturer's (EMD Chemicals) instructions.…”

Hepatoprotective effects of Yi Guan Jian, an herbal medicine, in rats with dimethylnitrosamine-induced liver fibrosis

“…The amplified RT-PCR products were subjected to electrophoresis at 100 V in a 2% agarose gel (Invitrogen, Carlsbad, CA) for 30 min. The transcription levels of each gene were normalized against those of GAPDH mRNA using a semi-quantitative method (Chen et al, 2006;Liu et al, 2008).…”

Porcine lactoferrin as feedstuff additive elevates avian immunity and potentiates vaccination

“…The sera were diluted up to 4 9 10 3 -fold for IgA detection and 8 9 10 4 -fold for IgG detection in the sample diluent solution from the kit, and absorbance was recorded at 450 nm by use of an ELISA plate reader (Liu et al 2008). The IBDV-specific antibody titer was determined by ELISA kit (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD), according to the manufacturer's instructions.…”