Development of a compact anti-BAFF antibody in Escherichia coli

Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichia coli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110 kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human.     

Construction and characterization of single-chain antibodies against human insulin-like growth factor-I receptor from hybridomas producing 1H7 or 3B7 monoclonal antibody

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.     

Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector

Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters

Tn-antigens are tumour-associated carbohydrate antigens that are involved in metastatic processes and are associated with a poor prognosis. MLS128 monoclonal antibody recognizes the structures of two or three consecutive Tn-antigens (Tn2 or Tn3). Since MLS128 treatment inhibits colon and breast cancer cell growth [Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends 3, 32-37.], understanding the interaction between MLS128 and Tn-clusters may allow us to the development of novel cancer therapeutics. Although MLS128 was previously reported to have specificity for Tn3 rather than Tn2, similar levels of Tn2/Tn3 binding were unexpectedly observed at 37°C. Thus, thermodynamic analyses were performed via surface plasmon resonance (SPR) using synthetic Tn2- and Tn3-peptides at 10, 15, 20, 25 and 30°C. SPR results revealed that MLS128's association constants for both antigens were highly temperature dependent. Below 25°C MLS128's association constant for Tn3-peptide was clearly higher than that for Tn2-peptide. At 30°C, however, the association constant for Tn2-peptide was higher than that for Tn3-peptide. This reversal of affinity is due to the sharp increase in K(d) for Tn3. These results were confirmed by NMR, which directly measured MLS128-Tn binding in solution. This study suggested that thermodynamic control plays a critical role in the interaction between MLS128/Tn2 and MLS128/Tn3.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Molecular cloning and characterization of a single-chain variable fragment antibody specific for benzoylecgonine expressed in Escherichia coli

Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding domain from the C1303 cells. Genes encoding an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, V_H) — linker — (light chain variable region, V_L)] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly₄-Ser)₃ was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432 bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific drug compounds or the detoxification of drug abusers by immunotherapy.     

Production of Anti-carbohydrate Antibodies by Phage Display Technologies: POTENTIAL IMPAIRMENT OF CELL GROWTH AS A RESULT OF ENDOGENOUS EXPRESSION*

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG₁ Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253–262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263–270). Similarly, anti-Le^x phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le^x)-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.     

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.     

Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system

We previously isolated phage antibodies from a phage library displaying human single chain antibodies (scFvs) by screening with a mannotriose (Man3)-bearing lipid. Of four independent scFv genes originally characterized, 5A3 gene products were purified as fusion proteins such as a scFv-human IgG1 Fc form, but stable clones secreting 1A4 and 1G4 scFv-Fc proteins had never been established. Thus, bacterial expression systems were used to purify 1A4 and 1G4 scFv gene products as soluble forms. Purification of 1A4 and 1G4 scFv proteins from inclusion bodies was also carried out together with purification of 5A3 scFv protein in order to compare their Man3-binding abilities. The present studies demonstrated that 1A4 and 1G4 scFv proteins have a higher affinity for Man3 than 5A3 scFv protein, which may determine whether scFv-Fc proteins expressed in mammalian cells are retained in the ER or secreted. Furthermore, the inhibitory effects of anti-Man3 1G4 scFv and anti-Tn antigen scFv proteins on MCF-7 cell growth were evaluated. Despite the fact that no obvious difference was detected in cell growth, microscopic observations revealed inhibition of foci formation in cells grown in the presence of the anti-carbohydrate scFv proteins. This finding provides a basis for the development of cancer therapeutics.     

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be ka = 4.03 x 104 M-1 s-1, kd = 5.77 x 10-4 s-1, KA = 6.98 x 107 M-1, and KD = 1.43 x 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.     

Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.     

Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange.

A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a 'diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

Coexpression of cancer-associated carbohydrate antigens,Tn and sialyl Tn

The expression of cancer-associated antigens, Tn and sialyl Tn, was examined using monoclonal antibodies, MLS 128 and MLS 102, recognizing these two antigens, respectively. A cell lysate from a human carcinoma cell line, LS 180 cells, was analysed by Western blotting using these two antibodies. Three glycoprotein bands were discernible with each antibody, of which two, corresponding to 250 and 210 kDa, were reactive with both the antibodies. LS 180 cells were metabolically labelled with³H-glucosamine and then the lysate from these cells was applied to two immunoaffinity columns. Sixty-five per cent of the Tn antigenic glycoproteins, based on radioactivity, bound to the MLS 102 affinity column. On the other hand, 45% of the sialyl Tn antigenic glycoproteins bound to the MLS 128 affinity column. These results indicate that some Tn and sialyl Tn antigens were expressed on the same polypeptide chains.The presence of non-sialylated GalNAc residues on the polypeptide chain with many Sia-GalNAc residues appears to be due to the incapability of three consecutive moieties of GalNAc-Ser/Thr to accept sialic acid.Abbreviations PSMF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - GalNAc N-acetylgalactosamine - Sia sialic acid     

From transcriptional landscapes to the identification of biomarkers for robustness

Background

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.

Results

The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.

Conclusions

Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.     

Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数