Contrastación de la hipótesis Secundarias

To enable the study of an enzymatic reaction, it is essential to have access to good quantities of pure protein. QPRTase has been isolated from a number of sources such as shiitake mushroom,1 _{castor bean endosperm}2 _{and mammalian liver and kidney.}3-5

However, such extractions are often very laborious, requiring several different chromatographic techniques to obtain the pure enzyme. Furthermore, isolation of sufficient protein for analysis requires access to significant quantities of the protein source. For example, 50 kg of shiitake mushroom were required to obtain sufficient QPRTase for analysis.

More recently, QPRTase from Salmonella typhimurium6

and Mycobacterium tuberculosis7_{were obtained by cloning the QPRTase gene into a suitable vector, then}

expressing the target protein in bacterial host cells. However, the methods employed for the purification of the over-expressed protein were still reasonably lengthy and required a number of steps involving different chromatographic techniques. Nevertheless, good quantities of pure protein were obtained which enabled kinetic and structural studies to be undertaken. The mechanistic studies that were carried out on the bacterial enzymes were discussed in sections 1.5 and 1.7.

Despite the studies on the bacterial enzymes, there were still many unanswered questions regarding the mechanism of the QPRTase catalysed reaction. Furthermore, although interest in QPRTase had been fuelled by the involvement of quinolinic acid in CNS disease,8 _{the human enzyme had yet to be studied. With this in mind, a}

project aimed at carrying out a thorough mechanistic study on human brain QPRTase was initiated in our laboratory in 2001.

The human QPRTase cDNA was isolated and characterised from a human brain DNA library by Fukuoka et al.9 _{The partial amino acid sequences for purified porcine}

kidney QPRTase were used to design oligonucleotide probes, which were screened against the human brain cDNA library. QPRTase was isolated as a single positive clone which encodes 1182 nucleotides and has a single open reading frame of 891 base pairs (291 amino acids). Confirmation that the isolated cDNA clone

It was found that introduction of the human cDNA into a QPRTase defective E. coli

strain brought about an abrupt increase in QPRTase activity and allowed the cells to grow in the absence of nicotinic acid. However, despite the enzyme being detected sufficient protein was not produced for isolation.

In our laboratory,10 _{using the cDNA for human brain QPRTase which was kindly}

donated by Professor S.I. Fukuoka (Research Institute for Food Science, Kyoto University, Uji, Japan), the human brain QPRTase gene was amplified by PCR. The expression vector pEHISTEV-QPRTase was then constructed by ligating the QPRTase gene into the pEHISTEV plasmid. Active human brain QPRTase was then successfully over-expressed in E. coli strain BL21 (DE3) host cells from the pEHISTEV-QPRTase construct.

A significant advantage of using the pEHISTEV plasmid is the high expression levels of the target protein. The pEHISTEV plasmid is comprised of 5365 base pairs, which includes a gene for resistance to the antibiotic kanamycin (Appendix 1). The DNA encoding human brain QPRTase was cloned into the multiple cloning site of the plasmid downstream from a T7 promoter. The expression of the QPRTase gene is therefore under the transcriptional control of the exceptionally strong T7 promoter. The transcription of a gene encoding T7 RNA polymerase in the host is controlled by the lac promoter and the lacoperator. A repressor is present which binds to the lac

operator and blocks transcription of T7 RNA polymerase. The repressor is a variant of thelac repressor and binds the operator more tightly than the wild-type repressor. This helps tighten regulation of transcription so that premature expression is prevented. Once the cells have reached the mid- to late-exponential stage of growth, the inducer isopropyl--D-thiogalactoside (IPTG) is added to bind the lac repressor and induce its dissociation from the lac operator, allowing expression of T7 RNA polymerase in the host, E. coliBL21 (DE3). The T7 RNA polymerase then binds to the T7 promoter and transcribes the QPRTase gene. Thereafter, the mRNA binds to the ribosome and is translated to QPRTase. As T7 RNA polymerase is highly active and extremely promoter-selective, the desired protein can make up more than 30% of the total cell proteins.

A further advantage of using the pEHISTEV plasmid is that protein purification is simple. When the expression vector is constructed, the target gene sequence is inserted adjacent to a short sequence encoding a 6-histidine tag and a TEV protease recognition site. As a result, the target protein is expressed with a polyhistidine affinity tag on the N-terminus and the protein can be easily purified by nickel affinity chromatography. The histidine-tag can then be selectively cleaved by digestion with TEV protease. TEV protease recognises a seven amino acid sequence Glu-X-X-Tyr-X-Gln-Gly, where X can be various amino acid residues. The sequence used in this study was Glu-Asn-Leu-Tyr-Phe-Gln-Gly, which is the most commonly used TEV protease recognition sequence (Appendix 1). Cleavage occurs selectively between the glutamine and glycine residues of the sequence. TEV protease is a cysteine protease and utilises a catalytic triad of residues (Cys-Asp-His) to catalyse peptide hydrolysis. Once the histidine tag has been cleaved from the target protein, a second nickel column can be used to isolate the pure protein.

This chapter describes the expression and purification of wild-type human brain QPRTase and the kinetic and structural studies that were carried out on the purified enzyme.