Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.
High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.
Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR 9025 CNRS-Institut de Biologie Structurale et Microbiologie, Marseille, France.
Ahmed
A
Aloulou
Philippe
P
Grandval
Josiane
J
De Caro
Alain
A
De Caro
Frédéric
F
Carrière
D002852:Chromatography, Ion Exchange; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008049:Lipase; C075987:pancreatic lipase-related protein 1