Molecular Modification Of Human Extracellular Superoxide Dismutase

Superoxide dismutase(SODs) are metalloenzyme playing an important role in protection.At present three distinct isoforms of SOD has been identified in mammals: SOD1 or human intracellular superoxide dismutase(Cu/Zn SOD,EC1.15.1.1),is found almost exclusively in intracellular cytoplasmic spaces;SOD2,or manganese superoxide dismutase(MnSOD,EC 1.15.1.1),exists in mitochondrial spaces;SOD3, or human extracellular superoxide dismutase(EC-SOD,EC 1.15.1.1),is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. Their specific activities are similar,and all can clear O₂^-·.EC-SOD is the only antioxidant enzyme that scavenges superoxide specifically in the extracellar compartment,which is detected in human plasma,lymph,ascites,and cerebrospinas fluids.EC-SOD scavenges superoxide anion,which limits oxidative stress and preserves nitric oxide bioabailability.EC-SOD has important biologic effects in tissues highly expressing EC-SOD including the vascular systerm and the lungs. EC-SOD protects against a numer of cardiovascular and lung diseases,attenuates brain injury,and preserves the regulation of erythropoietin in the hypoxic kidney.EC-SOD is a tetrameric secretory glycoprotein of molecular weight 135000 Da, with high affinity for heparin.Each subunit has one coper and one zinc atom.The primary structure of EC-SOD contains three parts.The N-terminal region supports the quaternary structure and contains the consensus for N-linked glycosylation at Asn89, which maintain the solubility of the protein.The C-terminal region mediates the binding of EC-SOD to components in extracellular matrix.The central region of human EC-SOD has the activity site and is approximately 50%identical to the final two-thirds of Cu/Zn-SOD and retains enzymatic activity at extreme pH,elevated temperature,and high concentrations of urea and guanidinium chloride.The same gene sequence may be due to different forms of disulfide bonds,which leads to the formation of different proteins.The analysis showed that the human EC-SOD subunit exists in two forms each with a distinct disulfide bridge pattern.One form is enzymatically active(aEC-SOD).while the other displays no SOD-activity(i EC-SOD);aEC-SOD contains a disulfide bridge homologous to Cu/Zn-SOD, Cys107-Cyst189,thus supporting enzymatic activity,in addition it contains a disulfide bridge between Cys45 and Cys 190.The cysteine residue at position195 is free and does not participate in disulfide bridge formation.The inactive form contains disulfide bridges between Cys107 and Cys195,and between the two vicinal Cys189 and Cys190.The protein is too large for structure determination by NMR.Indeed difficulties in crystallization of EC-SOD caused probably by glycosylation lead to the failure in determination of EC-SOD's three-dimensional structure by X-ray crystallography.In this study,we reported the construction of new protein,which is named as S-ECSOD, comprising the central domain of hEC-SOD.The recombinant protein can be produced in large quantities in E.coli BL21(DE3),and showed a single band at about 16.7 KDa by SDS-PAGE.The specific activity of S-SOD was 473 U/mg,which was proved by activity stain.Unlike human EC-SOD,the recombinant displays a less physical resistance toward high temperature and pH extremes,while the recombinant protein was found to withstand high concentration of urea and guanidine hydrochloride.We use the technology of molecular to cut some amino acid residues and free sulfydryls of hEC-SOD's primary structure to molecular modification.Designing the suitable primers to acquire the objective gene and connects with pET28a(+). Recombinant plasmid is expressed in E.coli BL21(DE3).To obtain high levels of soluble expression,we optimize the conditions for recombinant protein expression.The results show that,when OD₆₀₀ is 0.5-0.6 at 37℃, IPTG is added and final concentration achieve to 0.4 mM.After induction about 4h, protein is expression highly.This is the best conditions of expression.The Purification of the recombinant protein is through Ni-NTA,HiTrap Heparin HP and SP Sepharose Fast Flow.After three-step purification,the purified protein by SDS-PAGE electrophores is showed a single band,the size of 16.7 KD,and with negative activity by NBT staining,the bright band appears to verify the recombinant enzyme SOD activity.At the same time,the thermal stability of recombinant enzyme,pH tolerance and the stability of denaturant were measured and analyzed.Experiments showed that the recombinant enzyme has the better stability of a low-temperature.20℃and 40℃heat 90 min.still has 50%of the activity:however,in the 80℃heat insulation 15 min,the activity disappeared;good alkali resistance and acid-resistant less,pH 4.0,the activity is relatively low,only 35%of the original;as denaturant such as urea and guanidine hydrochloride concentration,enzyme activity increased.