REDES INSTALADAS ACTUALMENTE EN LA REGIÓN

The main aim and focus o f the present work w as to devise a system capable o f producing m illigram am ounts o f active P D E lA l for structural studies, to establish m ethods for purification o f recombinant P D E lA l and relate the biological activity o f the recombinant enzym e with the native dog heart P D E IA enzym e. Evidence suggesting that the dog heart P D E lA l cD N A encoded a functional gene w as given by sequence alignm ent studies o f the translated putative polypeptide am ino acid sequence compared to the bovine and human P D E lA l sequences. This alignm ent study show ed a strong hom ology (as discussed in section 1.8.2.1) suggesting that dog heart P D E lA l cD N A encoded for a functional gene.

The present work w as divided into tw o main sections. The first com prised o f the cloning and expression studies on the dog heart P D E lA l to find the m ost suitable host for the expression o f soluble recombinant P D E lA l. The second part concentrated on the biochem ical characterisation o f the recombinant enzym e. These goals are introduced below .

1.9.1 Cloning and expression of dog heart PD E lA l

The cloning and expression work involved the cloning of the PD E lA l cDNA into different expression vectors and analysis of subsequent expression in these expression systems to

discover the most suitable system. Three different expression systems, spanning

prokaryotic to eukaryotic, were explored and these are discussed briefly below. They will be discussed in more detail in the individual chapters dedicated to each system.

1.9.1.1 Yeast expression system

The yeast expression system chosen was that employing Pichia pastoris as the host. P.

pastoris is a methylotrophic yeast which means that it utilises methanol as its sole carbon

and energy source (Ogata et a l, 1969). It offers two main advantages over the traditional

Saccharomyces cerevisiae host. Firstly, the promoter used by P. pastoris to transcribe

foreign genes is an alcohol oxidase promoter (AOXl) which is highly repressed under non­ methanol growing conditions maintaining an “expression-off’ state minimising expression of foreign proteins during cell growth phase. This is a distinct advantage if the protein is toxic, and the over-expression o f PDE may cause adverse effects. The second advantage that P. pastoris has over S. cerevisiae is that the former does not have a tendency to ferment. A product of fermentation is ethanol which can rapidly build up to toxic levels in high-density cultures and cause cell death. Another advantage of the P. pastoris system is the option to export the recombinant protein out of the cell into the culture media. Therefore, P. pastoris offers itself as a useful host for the expression o f foreign proteins allowing scale-up of the expression system from shake-flasks to large-volume fermenter

vessels. The major drawback of the P. pastoris expression system is the requirement for

different media needed during the cell-growth phase and the protein expression phase.

In the present work, the P. pastoris expression system was set up for the first time in this laboratory and optimised so that it could be used for the expression of P D E lA l. The dog heart PD E lA l cDNA was cloned into the yeast vector pPICZaC. This vector had the signal sequence, a-Factor, for targeting expressed proteins towards a secretory pathway resulting in the secretion of proteins into the growth media. The advantage of secreted

part of the study was monitored by carrying out PDE activity assays on the culture media.

1.9.1.2 Bacterial expression system

Historically, Eschericia coli has been extensively used as a host for the expression of foreign proteins. It was the first organism to be used as a host for the production of foreign proteins. Many proteins have been successfully expressed using this host including PDEs, in particular PDE4 (Kovala et a l, 1997). E. coli requires relatively little manipulation for the expression of foreign proteins and is a relatively inexpensive host to use in terms of culture media required which are considerations when scale-up of the expression system is required. However, E. coli is unable to perform post-translational modifications such as glycosylation and phosphorylation, which are possible in a eurkaryotic host, and also necessary for many eukaryotic proteins. The production of co-factors and proteins important for assembly or stability may also be absent.

For the present work, dog heart PD ElA l cDNA was cloned into two different vectors. Firstly, it was cloned into the expression vector pGEX-3X. In this case, PD ElA l was produced as a fusion protein with the protein being N-terminal tagged with glutathione-S-

transferase (GST), a protein derived from the blood fluke Schistosoma japonicum. The

expression of soluble fusion protein was monitored using PDE activity assays as well as Western blot analysis using a commercial primary antibody raised against the GST. Since

E. coli are well known for producing inclusion bodies, especially when expressing foreign

genes toxic to the bacteria. Western blot analysis was also carried out on the insoluble material remaining following lysis of the bacteria. The second vector used for cloning the PD ElA l cDNA was pTrcHisA which produced an N-terminal histidine (His^) tagged PD E lA l protein. As before, detection of soluble PD E lA l was by carrying out PDE activity assays. For both expression vectors, different strains o f E. coli were explored to determine the effect, if any, of these strains on the expression of soluble P D E lA l.

1.9.1.3 Mammalian expression system

The final expression system explored was that using the Semliki Forest virus system. This system has been successfully used for the expression of many mammalian proteins

including thyroid peroxidase, dopamine receptor 3 and interleukin-4 (Blasey et a l, 2000). There are no reports of its use for the expression of PDEl enzymes. The advantage that it offers over the bacterial system is that it will process the target protein in terms of post- translational modifications as well as correctly fold the PD E lA l protein being expressed - both of which are necessary for the biological activity of the enzyme. However, protein expression using this system does require attention to biosafety considerations since there is a reported fatality from wild type SFV infection (Willems et a l, 1979). In vitro synthesis of PDEl A1 mRNA transcripts for transfection of the host cells (BKŒC-21) involves the use of commercially available biochemicals. The preparation of BHK-21 cells also requires training in the handling and processing of these cells in readiness for the transfection procedure. The details of the expression system are discussed later in the relevant chapter on this system (Chapter 5).

For the purposes of the cloning work for the Semliki Forest virus system, the full-length as well as N - and/or C-terminal truncated PDE lA l constructs were generated using PGR and then cloned into the vector pSFVl for expression in the Semliki Forest virus system. As before, expression of PDE 1A 1 was monitored using PDE activity assays as well as Western blot analysis using a commercial CaM-PDE antibody.

1.9.2 Characterisation of the soluble recombinant dog heart PDEIAI

The second part of the study involved the biochemical characterisation of the biologically

active recombinant PDEl A 1 enzyme to ascertain the K^, and IC50 values for

comparison with the native dog heart PDEl A purified by Clapham and Wilderspin (2001). The data obtained for the recombinant enzyme was also compared to published information regarding PDEl enzymes isolated from other tissues as well as other PDEs.

The native state o f the recombinant PDEl A 1 enzyme produced in the present study was also investigated using a combination of size exclusion chromatography and covalent cross- linking studies.

Chapter Two