Expression, Purification, And Characterization Of Rat Liver Mitochondrial Very-long-chain Acyl-CoA Dehydrogenase And G441Mutant-type

Objective-Develop a robust prokaryotic expression system to allow production of high quality and large quantities of wild-and mutant-type VLCAD protein. Through the expressed wild-and mutant-type VLCAD protein, we will able to study the basic biochemistry and investigate the structure-function relationship of this enzyme and its G441mutation site effect, thus facilitate advance our understanding of the clinical aspects of VLCAD deficiency caused by G441mutation.Methods-We used PCR reaction to subcloning rat liver mitochondrial VLCAD gene with polyhistidine-tag added on the oligonucleotide sequence construction. Then PCR products were identified using double restriction enyzme digestion and positive products were ligated into pLM1plasmid to facilitate protein expression. Single and double restriction enzyme was used to confirm the ligation product and positive clones were expressed using a prokaryotic E. coli expression system. A nickel ions held in chelated form on a chromatography affinity column was used to purify the expressed VLCAD protein.A QuikChange mutagenesis kit (Stratagene) was applied for constructing the mutant G441D and G441A VLCAD::pLM1expression plasmids, the following process was similar with the method use in expressed WT protein.To analyze expressed protein, we use the SDS-PAGE gel electrophoresis to evaluate the purity of protein expressed and Bradford method to determine protein concentration. Spectrophotometer was used to assay the enzymatic activity and kinetic studies toward palmitoyl-CoAs, and the enzyme substrate specificity toward various length carbons of acyl-CoAs.Results-We successfully subcloning VLCAD protein gene and recombinant WT protein produced in our study here had similar biochemical properties as previously report.Glycine residue was the most conserved residue revealed in our multiple alignment sequences analysis with frequency23.33%of glycine composition and35.89%among the entire conserved residue. A disease-causing missense mutation at G441site was found highly conserved in a Gly-Gly-X-Gly motif in various sources of VLCAD and other ACADs protein family. G441site were substituted into aspartic acid (G441D) and alanine (G441A), and both mutant-types proteins expressed showed similar characteristic with WT protein.Both the wild-and G441mutant-types protein expressed in yellow color after one-step purification, and the spectra analysis also showed a flavin-containing enzyme characterization. Protein assay indicated no significant different in amount expressed and no global conformational differences were revealed between the wild-and G441mutant-types protein.From the structural analyses, we predict G441plays an important role as a part of β-turns and located in a crucial position which is close to the FAD binding pocket and neighboring with several catalytic subunit residues. Our prediction showed mutation into G441D affects the β-turns structure, while mutation into G441A did not affect the β-turns.Nevertheless, substitution of amino acid G441into aspartic acid (G441D) and alanine (G441A) cause a significant loss of the enzymatic activity,11%and25%of WT protein activity, respectively. In addition, kinetic study also showed significant lower velocity reaction of both G441mutation-types compared with WT protein and the KM was3-4times higher for the mutant-types protein, indicating a higher saturation concentration for the optimum substrate is needed compared to the WT protein.Conclusions-1. Using a suitably tagged recombinant protein and an appropriate chromatographic medium, VLCAD protein can be expressed in prokaryotic expression system and a successful purification with high quality can be achieved in a single chromatographic step.2. G441is highly conserved in Gly-Gly-X-Gly motif in VLCAD and other ACADs protein family and play an important role in structure maintaining and functionally. The co-varying network of G441residue both structure maintaining and function-related positions were suggested.