Heterologous expression of three enzymes forstructural studies

This thesis work was aimed at the expression, purification and crystallization of three human proteins (bile acid CoA:amino acid N-acyltransferase, BAAT; farnesyl cysteine-carboxyl methyltransferase, Ste14; and stearoyl-CoA desaturase, SCD) in order to determine their three-dimensional structure using X-ray crystallography. The cDNA sequences were cloned into specific vectors for overexpression in heterologous systems. The choice of the best expression system was made considering the protein yield, stability and integrity before scaling up. Human BAAT was expressed in E.coli. The recombinant protein was purified by IMAC, reverse IMAC and gel filtration. The integrity of the sample was assessed by mass spectrometry and its homogeneity using DLS. The enzymatic activity was controlled verifying the presence of the products by mass spectrometry. In addition to the wild type protein, the catalytic mutant (C235A) was cloned and expressed to carry out crystallization trials in the presence of the substrate. The samples obtained are sufficiently pure, stable and homogeneous to set up crystallization trials. Crystals were obtained only with the mutant but they are not sufficiently ordered for X-ray diffraction experiments. Using the high-throughput system published by Drew et al., 2008, the membrane protein Ste14 from C.glabrata was selected as a good candidate for a large scale production in order to conduct structural studies. The system uses S.cerevisiae to overexpress membrane proteins; the protein of interest is cloned into a GFPfusion vector allowing to estimate the expression level and the stability in several detergents by measuring fluorescence directly in cell extracts. The enzyme was purified by IMAC, reverse IMAC and gel filtration in the presence of the detergents DDM and LDAO. The gel filtration profile indicates that the sample is ‘monodisperse’ and hence adequate to set crystallization trials. Up to now no suitable crystals were obtained. Finally, a protocol for the expression and purification of the human membrane enzyme SCD was developed. The recombinant protein was expressed in insect cells and purified in the presence of the detergent DDM by IMAC. Until now the yield and the purity obtained are not optimal but a sufficient quantity of the enzyme can be extracted to carry out crystallization trials aimed at structural and biochemical studies.