title: 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 x mg protein(-1) x 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 x L(-1) vs 36 mg x L(-1)) and total enzyme activity increased about 5 folds (1900 IU x L(-1) vs 367 IU x 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.
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Chen
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Jiayong
authors.initials: J
authors.lastname: Tang
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Jia
authors.affiliation: Chongqing Academy of Animal Science, Chongqing, 402460, China.
authors.forename: Dingbiao
authors.initials: D
authors.lastname: Long
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Guangmang
authors.initials: G
authors.lastname: Liu
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Chen
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Shang
mesh_terms: D000818:Animals; D003062:Codon; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D034421:Sus scrofa / Q000235:genetics*; D019076:Transgenes
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D008049:Lipase