Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) complexed to protein dis ulphide 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 th ose 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 necessa ry to produce milligram quantities of pure protein. We chose the baculoviru s expression system for this purpose. Insects cells were co-infected with r ecombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulti ng heterodimer was purified by affinity chromatography. From 5 litres of in sect 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% b eta-structure content. The recombinant protein had a comparable triglycerid e 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 m ethodology for the purification of the resulting protein complex.