Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Abstract

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol.mg protein −1.min−1) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L−1 vs 36 mg.L−1) and total enzyme activity increased about 5 folds (1900 IU.L−1 vs 367 IU.L−1) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.

Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

“…After 3 days induction by 0.5% methanol, P. pastoris cells were collected and applied for total RNA extraction. Total RNA extraction and quality control, qPCR procedure, and relative gene abundance quantification were the same as previously described by our group [24]. The relative gene expression was analyzed using qPCR (ABI7900HT, Applied biosystem).…”

“…In the present study, novel hybrid protein combining PI with hLY was designed. Considering codon optimization facilitates the recombinant expression of many heterogeneous proteins or AMPs derived from various sources [24,40,41], we firstly optimized the coding sequence of rehLY-PI with biased codons of P. pastoris and successfully constructed the P. pastoris rehLY-PI/pPICZaA transformant. The production of recombinant rehLY-PI reached 86 ± 12.5 mg/L after 96 h induction with 0.5% methanol in a shaking flask.…”

“…Therefore, it is necessary to efficiently express recombinant antimicrobial peptide parasin I fused with human lysozyme in yeast expression system. It is reported that heterogeneous protein expression level in P. pastoris strongly depends on the biased codon usage [24]. However, producing a functional hLY-PI hybrid protein by fusing the PI and hLY into a single gene has not been previously reported.…”

Cloning, expression, and characterization of a porcine pancreatic α-amylase in Pichia pastoris

“…Escherichia coli TOP10 (Invitrogen, Beijing, China) was used for plasmid amplification. The plasmid pPICZαA (Invitrogen, Beijing, China) was used for the production of His-tagged PPA proteins, and the P. pastoris X-33 strain (Invitrogen, Beijing, China) was used as the protein expression host ( Zhao et al., 2014 ). The E. coli TOP10 strain was grown in LB medium at 37 °C and P. pastoris X-33 strain was grown in yeast extract-peptone-dextrose medium at 28 to 30 °C ( Zhao et al., 2014 ).…”

“…The plasmid pPICZαA (Invitrogen, Beijing, China) was used for the production of His-tagged PPA proteins, and the P. pastoris X-33 strain (Invitrogen, Beijing, China) was used as the protein expression host ( Zhao et al., 2014 ). The E. coli TOP10 strain was grown in LB medium at 37 °C and P. pastoris X-33 strain was grown in yeast extract-peptone-dextrose medium at 28 to 30 °C ( Zhao et al., 2014 ). The AMV reverse transcriptase, T4 DNA ligase, Taq DNA polymerase, pMD18-T vector, restriction enzymes ( Xba I, Knp I, Sac I), DL2000 DNA marker, protein marker were purchased from TaKaRa (Dalian, China).…”