Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

D1蛋白酶的克隆表达、纯化、生物活性测定及多克隆抗体的制备

为了开发以D1蛋白酶作为靶标的新型除草剂,需要对先导化合物的生物活性进行检测和筛选。从菠菜中提取总RNA,逆转录合成cDNA,采用PCR扩增了CtpA的基因,连接至表达载体pET-28a中,构建了重组表达质粒,并在大肠杆菌BL21(DE3)中进行高效表达,通过降低IPTG和诱导温度获得了可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析和凝胶过滤柱层析对重组蛋白进行了纯化,SDS-PAGE和Western blotting结果证实目的蛋白为含有His-tag的融合蛋白。以合成的D1前体蛋白羧基端24肽作为模拟底物,采用高效液相色谱法测定了其水解活性,结果表明活性可达1.10nmol/(mg·min),为文献报道数据的15倍。纯化后的CtpA蛋白免疫日本的长耳大白兔制备多克隆抗体,ELISA法测定其血清抗体的效价高达1:100000。该结果为抑制剂先导化合物的筛选和酶蛋白与化合物的作用机理研究提供了必要的基础。     

Removal of catalytic activity by EDTA from antibody light chain

Gp41 peptide antigen of the HIV-1 envelope (TP41-1:TPRGPDRPEGIEEEGGERDR, a highly conserved region) was enzymatically degraded by the antibody light chain 41S-2-L after an induction period. The peptide bond between Glu¹⁴ and Gly¹⁵ was cleaved early in the reaction. When EDTA was added in the induction period, it inhibited the degradation of TP41-1 thus ceasing the catalytic activity of 41S-2-L. In contrast, when EDTA was added after the induction period, only a small reduction in the catalytic activity was observed. These observations suggest that metal ions are important in stimulating catalytic activity early in the reaction.     

Molecular simulation studies of a selenium-containing scFv catalytic antibody that mimics glutathione peroxidase

GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency.In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme–GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.     

Chemical modification of a catalytic antibody that accelerates the hydrolysis of carbonate esters

Catalytic antibody, 4A1, catalyzes the hydrolysis of p-nitrophenyl alkyl carbonate. To determine the amino acid residues related to the catalytic activity of the antibody, we studied the effect of Tyr-, Trp-, and Lys-selective reagents on the catalytic activity and determined the amino acid sequences around the modified amino acid residues. We found that the Tyr-selective reagent is the most effective one and the modification of one Tyr residue results in the complete loss of the catalytic activity. The modified Tyr residue is identified to be Tyr-32 in the CDR-1 of the L chain.     

Temperature and the catalytic activity of enzymes: A fresh understanding

The discovery of an additional step in the progression of an enzyme from the active to inactive state under the influence of temperature has led to a better match with experimental data for all enzymes that follow Michaelis–Menten kinetics, and to an increased understanding of the process. The new model of the process, the Equilibrium Model, describes an additional mechanism by which temperature affects the activity of enzymes, with implications for ecological, metabolic, structural, and applied studies of enzymes.     

Isolation of novel catalytic antibody clones from combinatorial library displayed on yeast-cell surface

A combinatorial library of the Fab fragment of a catalytic antibody able to hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol was constructed on yeast-cell surface. Interesting clones were selected using fluorescence-activated cell sorting (FACS). When binding affinity to a transition-state analog was detected, evolution of the catalytic antibody was carried out in vitro on yeast-cell surface. A number of variants with enhanced catalytic activity and binding affinity were obtained. The results showed that the improvement of catalytic antibody, which can be performed easily on yeast-cell surface using the cell-surface engineering system, is a good example of the application of protein library construction.     

Mixed-ligand thiosemicarbazone complexes of nickel: Synthesis, structure and catalytic activity

Reaction of thiosemicarbazones of salicylaldehyde, 2-hydroxyacetophenone and 2-hydroxynaphthaldehyde with Ni(ClO₄)₂·6H₂O, using 2,2′-bipyridine as coligand, afforded three dinuclear complexes (1a, 1b and 1c). Similar reactions using 2,2′:6′2″-terpyridine as coligand yielded three mononuclear complexes (2a, 2b and 2c). Crystal structures of 1b and 2a have been determined. In the dinuclear complexes, one nickel center is surrounded octahedrally by a dianionic O,N,S-donor thiosemicarbazone, a bipyridine and the bridging phenolate oxygen of the other thiosemicarbazone. The second nickel center adopts a square-planar geometry created by the second O,N,S-coordinated thiosemicarbazone and the bridging sulfur of the first thiosemicarbazone. In the mononuclear complexes nickel is complexed by a monoanionic O,N,S-coordinated thiosemicarbazone and a terpyridine, and the cationic species are isolated as perchlorate salts. All these six complexes are paramagnetic (μ_eff = 2.63-2.92 B.M.) and in dimethylsulfoxide solution they show intense absorptions in the visible and ultraviolet region, origin of which has been probed through DFT calculations. Cyclic voltammetry on the complexes shows one irreversible oxidation of coordinated thiosemicarbazone on the positive side of SCE, and one irreversible reduction of the coordinated polypyridine ligand on the negative side. These nickel complexes are found to be efficient catalysts for Suzuki cross-coupling reactions.     

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38 U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55 °C and 50 °C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60 °C and apparent melting temperature of reBaxA50 were 9.74 min and 89.15 °C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.     

定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性

本研究旨在利用理性设计的方法来提高来源于土曲霉Aspergillus terreus的酸性脂肪酶ATL的催化活力。通过同源比对,选择脂肪酶盖子区域和底物结合口袋域中的位点进行定点突变,得到8种ATL的突变脂肪酶。结果发现,盖子区域突变酶ATLLid与底物结合口袋域突变酶ATLV218W的催化活性显著提高。ATLLid和ATLV218W对底物对硝基苯酚月桂酸酯p-nitrophenyl laurate(p-NPL)的催化活性最高,k_(cat)值较ATL分别提高了39.37倍和50.79倍,k_(cat)/K_m值较ATL分别提高了2.85倍和8.48倍。与ATL相比,ATLLid和ATLV218W的热稳定性略有下降,最适p H为5.0,p H 4.0–8.0具有较好的稳定性,说明突变未对ATL的嗜酸耐酸特性产生影响。通过同源建模模拟及分子对接技术分析底物p-NPL与酶分子间的相互作用,解析了ATLLid和ATLV218W催化活性提高的机理。     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

N-terminal deletion affects catalytic activity of saporin toxin

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated proteins with deletions at the N-terminus and at the C-terminus. An N-terminal (sapDelta1-20) deletion mutant of saporin displayed defective catalytic activity, drastically reduced cytotoxicity but increased ability to interact with liposomes inducing their permeabilization at low pH. A C-terminal (sapDelta239-253) deletion mutant showed instead a moderate reduction in cytotoxic activity. A substantial alteration of secondary structure was evidenced by Fourier transformed infrared spectroscopy (FTIR) in the sapDelta1-20 mutant. It can be hypothesized that the defective functions of sapDelta1-20 are due to alterations of its spatial configuration.     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency

The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.     

Design, synthesis and catalytic activity of a serine protease synthetic model

The design, synthesis and catalytic properties of acyclic branched peptide carrier that possesses thecatalytic triad residues of the serine proteases isreported. The synthesis of the peptide model wastotally completed on solid support using threedifferent orthogonal amino protecting groups.Hydrolytic activity measurements againstSuc-Ala-Ala-Ala-pNA substrate showed that it ishydrolysed by the peptide model to a small extent.Despite this small hydrolytic activity, it is thefirst time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound.Contrary, this enzyme model peptide showedconsiderable activity against the Boc-Ala-pNPsubstrate (k_cat = 0.414 min^–1 and K_m = 0.228 mm). These results suggest that thedesigned carrier brings in appropriate contact thecatalytic triad residues (Ser, His, Asp) resulting inthe obtained hydrolytic activity.     

Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.     

Design, synthesis and catalytic activity of a serine protease synthetic model

Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model peptide showed considerable activity against the Boc-Ala-pNP substrate (k _cat =0.414 min⁻¹ andK _m =0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser, His, Asp) resulting in the obtained hydrolytic activity.