title: High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris.
abstract: The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.
authors.affiliation: INSERM 476-INRA 1260-Univ Aix-Marseille, 27 Bd Jean Moulin, Marseille, F-13385, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Sebban-Kreuzer
authors.forename: Paule
authors.initials: P
authors.lastname: Deprez-Beauclair
authors.forename: Amelie
authors.initials: A
authors.lastname: Berton
authors.forename: Isabelle
authors.initials: I
authors.lastname: Crenon
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D014415:Tunicamycin; D008049:Lipase; C075988:pancreatic lipase related protein 2