Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.
The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-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.
MRC Molecular Medicine Group, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK.
P J
PJ
Ritchie
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A
Decout
J
J
Amey
C J
CJ
Mann
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J
Read
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M
Rosseneu
J
J
Scott
C C
CC
Shoulders
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera
D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C106092:microsomal triglyceride transfer protein; D019704:Protein Disulfide-Isomerases