Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris

被引:19
作者
Aloulou, A [1 ]
Grandval, P [1 ]
De Caro, J [1 ]
De Caro, A [1 ]
Carrière, F [1 ]
机构
[1] Inst Biol Struct & Microbiol, UPR 9025 CNRS, Lab Enzymol Interfaciale & Physiol Lipolyse, F-13009 Marseille, France
关键词
constitutive expression; Pichia pastoris; heterologous protein expression; glyceraldehyde 3-phosphate dehydrogenase promoter; pancreatic lipase-related protein;
D O I
10.1016/j.pep.2006.01.004
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRPI) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRPI 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 HPLRPI 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. (c) 2006 Elsevier Inc. All rights reserved.
引用
收藏
页码:415 / 421
页数:7
相关论文
共 40 条
[1]   Pancreatic lipase-related protein 2 but not classical pancreatic Lipase hydrolyzes galactolipids [J].
Andersson, L ;
Carriere, F ;
Lowe, ME ;
Nilsson, A ;
Verger, R .
BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM, 1996, 1302 (03) :236-240
[2]   Molecular evolution of the pancreatic lipase and two related enzymes towards different substrate selectivities [J].
Carriere, F ;
Bezzine, S ;
Verger, R .
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 1997, 3 (1-4) :55-64
[3]  
Cregg JM, 1998, METH MOL B, V103, P27
[4]  
DATTA S, 1988, J BIOL CHEM, V263, P1107
[5]   ASSIGNMENT OF HUMAN PANCREATIC LIPASE GENE (PNLIP) TO CHROMOSOME 10Q24-Q26 [J].
DAVIS, RC ;
DIEP, A ;
HUNZIKER, W ;
KLISAK, I ;
MOHANDAS, T ;
SCHOTZ, MC ;
SPARKES, RS ;
LUSIS, AJ .
GENOMICS, 1991, 11 (04) :1164-1166
[6]  
De Caro A, 1999, METH MOL B, V109, P239
[7]   Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice [J].
De Caro, J ;
Sias, B ;
Grandval, P ;
Ferrato, F ;
Halimi, H ;
Carrière, F ;
De Caro, A .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 2004, 1701 (1-2) :89-99
[8]   Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species [J].
De Caro, J ;
Carrière, F ;
Barboni, P ;
Giller, T ;
Verger, R ;
De Caro, A .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1998, 1387 (1-2) :331-341
[9]  
DeLisle RC, 1996, EUR J CELL BIOL, V71, P62
[10]   Heterologous protein production in methylotrophic yeasts [J].
Gellissen, G .
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2000, 54 (06) :741-750