Reconstruction Of Selenium-containing Human Single-chain Abzyme With GPX Activity

Glutathione peroxidase (GPX) is an important member of antioxidative enzyme system in vivo, and is a kind of protein containing selenium. GPX can protect organisms from the damage coming from active oxygen species through scavenging hydrogen peroxide and other hydroperoxides. GPX has strong antioxidant ability, so it is important in treatment and prevention of many diseases. Due to the limitations associated with native GPX, such as instability, limited source, people have made a great deal of efforts to study the GPX mimics. The key of GPX mimics is the specific binding site to GSH. Abzyme is a kind of immunoglobulin with catalytic activity. It solves the problem of substrate binding sites perfectly. However, the initial abzyme acquired from the monoclonal antibodies has some drawbacks as drugs, such as the big molecular size and strong immunogenicity. In the past years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-life in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte. So the single-chain antibody is more suitable for using as drugs.In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. Based on the results of structural analysis, site-directed mutation was carried out for scFv-B3.We have carried out the following works in this study:1. We cooperate with group of Zheng from Theoretical Chemistry Research Institute of Jinlin University on structural analysis of scFv-B3, and two possible substrate binding sites was predicted——site1 and site2. Site 1 is mainly composed of nineteen residues, in which eleven residues are non-polar residues, and the other eight residues are polar residues. Site 2 is composed of eighteen residues, in which six residues are non-polar residues, and the other twelve are polar residues. After docking study, we calculated the interaction energies of the substrate with each of the residues in the active site of scFv-B3. it was determined that the total interaction energy in Site 1 is more negative than that in Site 2. It indicates that total interaction energy between site 1 and the substrate is lower, and their binding force is stronger, therefore, Site1 is the more likely binding site. Besides, there are three hydrogen bonds with Trp113, Gln174, and Gln179 in Site 1, compared to two hydrogen bonds with Gln175 and Asp222 in Site 2. Thus, these hydrogen-bonding interactions are stronger in Site 1 than in Site 2, therefore, the combination between the substrate and site 1 will be more stable.In the reactions catalized by GPX, catalytic group works through the sulfhydryl of GSH. Accordingly, research of molecular docking was carried out to analyse the distance between the GSH and every amino acid of the two sites. In both the pockets predicted previously no serine was found, and the serines surrounding the pocket may be too far from the sulfhydryl of GSH, possibly explaining why the Se-scFv-B3 has lower catalytic activity. We wished to find one amino acid in each presumed binding site to be mutated to Ser. We investigated the polar, geometric, and the distance properties of each amino acid with GSH, and chose the candidates following according to a number of criteria. First of all, each candidate amino acid must not be one of the key residues comprising and maintaining the conformation of the site, otherwise the mutant would destroy the conformation of the site. Secondly, the candidate amino acid should be comparable with Ser with regards to molecular size and properties. Finally, Ala180 in Site 1 and Ala44 in Site 2 were the optimal choices for the candidate amino acid.2. In this study, the QuikChange sitedirected mutagenesis method was used to transform Ala180 and Ala44 into Ser ,according to structural analysis of scFv-B3. Using the plasmid pPelB-B3 as the template, synthesize two complimentary oligonucleotides containing the desired mutation, extend during temperature cycling by PfuTurbo DNA polymerase, incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Then the product is treated with Dpnâ… . The Dpnâ… endonuclease (target sequence: 5′-Gm6ATC-3′) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA. Synthetical DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpnâ… digestion. The nicked vector DNA containing the desired mutations is then transformed into Rosetta competent cells. Plasmid of the Recombinants was sequenced to obtain the mutants——scFv-B3-A44S and scFv-B3-A180S. The two mutants,scFv-B3-A44S and scFv-B3-A180S, were expressed as soluble proteins in E. coli Rosetta together with scFv-B3, and purified by immobilized Ni²⁺ chelation chromatography. We have successfully purified the interesting scFv with 150 mM imidazole.。The results of SDS-PAGE showed that their molecular masses were about 31 kDa, consistent with calculations. Western blot showed that the wild type scFv-B3 and the two mutants were capable of binding to monoclonal antibody against hexa-histidine, further indicating that the three scFvs were expressed and purified successfully.3. GPX plays an important role in anti-oxidation using GSH as the reducing substrate and selenocysteine(Sec)residues as catalytic group. Se-scFvs were prepared by chemically modifying the hydroxyls of reactive serines of antibodies. The Se-containing scFvs, including mutants and the wild, all showed GPX activity. However, the activity of Se-scFv-B3-A180S was determined to be 2646U/mmol, approximately double that of the previously studied Se-scFv-B3 demonstrating that the previous structural and active sites analysis correct. We determined the selenium content of all Se-containing scFvs and found that there are five seleniums in Se-scFv-A180S, compared with four in Se-scFv-B3, that means the activity of Se-containing abzyme is benifited from many Secs, it also explained the reason why Se-scFv-B3 with GPX activity before mutant. At present, the problem is that the production is very low, maybe related to long-term preparation, or the composition of amino acids of the antibody. We will strive for more in-depth research in this area in the future.