Studies On Expression Of HCu, Zn-SOD In Pichia Pastoris

Superoxide dismutase, which catalyzes the dismutation of the superoxideradical to molecular oxygen and hydrogen peroxide are found in all free-livingorganisms except some oxygen-sensitive obligate anaerobes. Superoxide radicalis a one-electron reductive product of molecular oxygen, and is generated as aby-product of oxidative metabolism. The family of superoxide dismutase hasCu and Zn, or Mn, or Fe at the active site which is responsible for the reaction:2 O 2-+ 2H+→ H2O2 + O2Cu,Zn-SOD exhibits an unusual structural stability and a strong dimericinteraction. Bovine SOD has a conformational melting temperature of 83℃ andis apparently the most thermally stable among the currently characterizedglobular protein. The dimmer is composed of two identical subunits, each ofwhich has 151 amino acids. The skeletal structure of SOD is an eight-strandedβbarrel with an external pair of double-stranded loops that form the active sitechannel. The subunit topology is that of a Greek key βbarrel similar toimmunoglobulin domain.As superoxide radicals are involved in the pathogenesis of a variety ofdiseases,more and more attentions are paid into the clinical application of SOD.Human Cu, Zn-SOD has been shown to be effective for treating inflammatorypathologies, reducing reperfusion injuries, and decreasing blood pressure inmodel animal. HSOD has recently received orphan drug status for theprevention of pumonary dysplasia in premature infants.In 1999, we obtained the cDNA encoding human Cu, Zn-SOD whichwas derived from the human liver by PCR and sequenced. The 0.49kb DNAfragment obtained was cloned into pBV220 vector to construct a recombinantplasmid designated pBVSOD. Results from SDS-PAGE show that a specificprotein with a molecular weight of 19KD ,it is at a level of 30.17 % of the totalbacterial proteins appears in E.coli harboring pBVSOD, which is absent incontrol bacteria harboring pBV220.However, the use of human Cu,Zn-SOD in human medicine is highlylimited because of the short half-life due to kidney clearance. Endothelial cellsare highly enriched with acidic proteoglycans such as heparan sulfate. Proteinswith high affinity to heparin-like proteoglycans were found to localize on theouter surface of vascular endothelial cells. The binding to the negativelycharged molecular heparin and heparin sulfate is of electrostatic nature.Caldwell, E.E.O.,et al.obtained a heparin-binding peptide R7 which eluted at0.82 M NaCl on heparin sepharose column by the selection from a randomcombinatorial library of 7-mer peptides. Therefore we choose a strench of R7 asthe heparin-binding domain.To minimize possible disturbance, a flexible linkerof GPGLWGGGGGGG was selected and GP GLWG was homologous tothe196-173 part of extracellular superoxide dismutase.The heparin-binding peptide gene fused into the C-terminal ofCu,Zn-SOD. The fused gene was ligated into pPIC 9 vector designated pPIC 9HBSOD. The recombinant vector permitted the expression of HBSOD at up to10.3% of total Pichia pastoris protein . Estimation of the molecular weight bySDS-PAGE confirmed that HBSOD molecular weight was increased by theamount (21800D) expected for the addition of the heparin-binding peptide.HBSOD were purified through the procedures of heat denaturation,DEAE-Sepharose F.F.,Sepharyl S-100 H.R.. The HBSOD has normal activityof SOD. However, surface charge changes may affect the optimum pH ofHBSOD making it shift to alkaline range . It is known that surface chargesdistribution of enzyme produce ionic environments or electrostatic fields whichcan stabilize or destabilize the polar group embedded inside the molecular.Arg143 at the active site of SOD maybe stabilized by the acidic amino acids ofthe surface. The addition of 7Arg may disturbe the electrostatic field and morebasic solution can offset the influence. On the other hand, the more positivelycharged surface of HBSOD make it more easily contact with the negativelycharged membrane of cells which is benefit for the clinical application. HBSODis more thermo stable than SOD. The addition of the heparin-binding peptideimproves the hydrophilic/hydrophobic ratio of the native enzyme. This changemakes it more difficult for the hydrophobic group to access to the solution. Asthe interaction of the two subunits is important for the stability, it is suggestedthat the heparin-binding peptide may not disturbed the stereo structure.The distinguish characters of our work are that we uses the results ofselection of heparin affinity from a random combinatorial library. The selectedpeptide has not only high affinity for heparin, but also small molecular weightwhich maybe important for functional units interaction. In addition, thepositively charged residues would neutralize some of the negative chargesfound on the native Cu, Zn-SOD that may improve its close contact with cellsurfaces or its equilibration between vascular and endothelial cell surfaces. Weare investigating other possibilities, including the strategies of directionevolution, to improve the pharmacological properties of human superoxidedismutase.