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Titolo:
Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein
Autore:
Ritchie, PJ; Decout, A; Amey, J; Mann, CJ; Read, J; Rosseneu, M; Scott, J; Shoulders, CC;
Indirizzi:
HammersmithEnglandImperial Coll Sch Med, MRC, Mol Med Grp, London W12 0NN,Hammersmith Hosp London England W12 0NN RC, Mol Med Grp, London W12 0NN, State Univ Ghent, Dept Biochem, Lab Lipoprot Chem, B-9000 Ghent, Belgium State Univ Ghent Ghent Belgium B-9000 poprot Chem, B-9000 Ghent, Belgium
Titolo Testata:
BIOCHEMICAL JOURNAL
, volume: 338, anno: 1999,
parte:, 2
pagine: 305 - 310
SICI:
0264-6021(19990301)338:<305:BEABCO>2.0.ZU;2-A
Fonte:
ISI
Lingua:
ENG
Soggetto:
B-CONTAINING LIPOPROTEINS; DISULFIDE-ISOMERASE; APOLIPOPROTEIN-B; ABETALIPOPROTEINEMIA GENE; ESCHERICHIA-COLI; COMPLEX; RESOLUTION; SECRETION; SUBUNIT; IDENTIFICATION;
Keywords:
apolipoprotein B; endoplasmic reticulum; lipoprotein; protein disulphide isomerase; protein purification;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
30
Recensione:
Indirizzi per estratti:
Indirizzo: Shoulders, CC HammersmithEnglandImperial Coll Sch Med, MRC, Mol Med Grp, London W12 0NN, Hammersmith Hosp London England W12 0NN p, London W12 0NN,
Citazione:
P.J. Ritchie et al., "Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein", BIOCHEM J, 338, 1999, pp. 305-310

Abstract

The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons andvery-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease, To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95 % pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34 % alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 26/09/20 alle ore 23:38:03