共查询到20条相似文献,搜索用时 31 毫秒
1.
Activation of a Refolded, Berberine-specific, Single-chain Fv Fragment by Addition of Free Berberine
A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (VH) and light chain (VL) genes were connected with a flexible linker using an assembly PCR. The VH-linker-VL gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine. 相似文献
2.
Sergey M. Kipriyanov Stefan Dübel Frank Breitling Roland E. Kontermann Stefan Heymann Melvyn Little 《Cell biochemistry and biophysics》1995,26(3):187-204
Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase
II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus.
After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by
size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly
of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation
procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by
enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays
showed that covalently linked (scFv′)2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives,
specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot
analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection. 相似文献
3.
John de Kruif Arjen Kramer Therèse Visser Carina Clements Roy Nijhuis Freek Cox Vanessa van der Zande Renate Smit Daniel Pinto Mark Throsby Ton Logtenberg 《Journal of molecular biology》2009,387(3):548-4235
To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (VH) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (VL) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the VH regions in this VL-restricted panel with a previously published repertoire of anti-TT VH regions with cognate VH-VL pairing showed a very similar distribution of VH, DH and JH gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-VL anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-VL anti-TT VH repertoire was combined with a collection of naïve VL regions and again selected for binding to TT, many of the VH genes were recovered in combination with a diversity of VL regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT VH combined with 15 diverse VL chains were determined and found to be identical to each other and to the original isolate restricted to a single-VL chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding VH regions from an immunized repertoire have promiscuous features. These VH regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, VH region diversity and affinity of a natural repertoire. 相似文献
4.
Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin
(CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in
IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine
residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding
while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue
at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine
residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are
important in determining their ability to bind CL. 相似文献
5.
《MABS-AUSTIN》2013,5(6):1058-1071
Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability. 相似文献
6.
Andreas Lehmann Josephine H F Wixted Maxim V Shapovalov Heinrich Roder Roland L Dunbrack Jr. Matthew K Robinson 《MABS-AUSTIN》2015,7(6):1058-1071
Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability. 相似文献
7.
K. Reed Clark Scott T. R. Walsh 《Protein science : a publication of the Protein Society》2009,18(12):2429-2441
We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (VH) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the VH domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (VL) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water. 相似文献
8.
We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis. A combinatorial library of 107 clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay. Selected VL and VH sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab. Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.Key words: phage display, antibody maturation, Fab, antibody folding, scFv, agglutination 相似文献
9.
Geovanny Rivera-Hernández Marta Marín-Argany Bernat Blasco-Moreno Jaume Bonet Baldomero Oliva Sandra Villegas 《MABS-AUSTIN》2013,5(5):678-689
Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ1–42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest. 相似文献
10.
Jean‐Luc Pellequer Shu‐wen W. Chen Victoria A. Roberts John A. Tainer Elizabeth D. Getzoff 《Journal of molecular recognition : JMR》1999,12(4):267-275
We have analyzed conformational changes that occur at the interface between the light (VL) and heavy (VH) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three‐dimensional structures to evaluate changes in compactness of the VL–VH interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium‐sized nonprotein antigens cause an opening of the VL–VH interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the VL–VH interface. The largest changes in the atomic buried contact surface area at the VL–VH interface occur in residue pairs providing two ‘shock absorbers’ between the edge β‐strands of the VL and VH β‐sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the VL–VH interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the VL–VH interface can be used to change antibody binding affinity with antigens. Due to the tightly packed VL–VH interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity. Copyright © 1999 John Wiley & Sons, Ltd. 相似文献
11.
Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the VH and VL gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (KD =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein. 相似文献
12.
Shovanlal Gayen 《FEBS letters》2010,584(4):713-718
The C-terminal residues 98-104 are important for structure stability of subunit H of A1AO ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (HI93C, HL96C and HL98C). Together with the surface charge distribution of H85-104, these results shine light into the A-H assembly of this enzyme. 相似文献
13.
Crystal N. Falco Kaitlyn M. Dykstra Bradley P. Yates Peter B. Berget Professor 《Biotechnology journal》2009,4(9):1328-1336
Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors. 相似文献
14.
Yoshiaki Iwasaki Hiroyuki Takabatake Marc Monestier Soldano Ferrone 《Immunogenetics》1995,42(2):90-100
The anti-HLA-DQ3 monoclonal antibodies (mAb) KS13, SO1, SO2, SO3, SO4, and SO5 recognize spatially close but distinct antigenic determinants, since they crossinhibit each other in their binding to HLA-DQ3 antigens, but do not share idiotopes recognized in their antigen combining site by syngeneic and anti-id antisera and mAb. Furthermore, mAb SO1, SO3, SO4, and SO5 react also with HLA-DQ allospecificities other than HLA-DQ3. Sequence analysis of the heavy (V
H
) and light (V
L
) chain variable region of the six mAb revealed preferential usage of V
H
36–60 and V
K
12/13 gene families. However, the individual V
H
and V
L
germline gene usage by the six mAb is diverse and the utilization of D, J
H
, and J
L
gene segments is heterogeneous. The diverse usage of V
H
and V
L
gene segments and heterogeneous amino acid sequences of V
H
and V
L
CDR, together with the heterogeneous idiotypic profile, may reflect the complexity of the determinants recognized by the six mAb on HLA-DQ3 antigens. The results we have presented provide for the first time information about the structural basis of the diversity of antibodies recognizing human histocompatibility antigens.The nucleotide sequence data reported in this Papershave been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L20499, L20957, L20961, L24557, L24558 and L20962, respectively, for V
H
region genes, and L20956, L20958, L24555, L24556, L20959, and L20960, respectively, for V
L
region genes 相似文献
15.
Haruo Suzuki Etsuko B. Mukouyama Chieki Wada Yasuko Kawamura-Konishi Yumiko Wada Mitsunori Ono 《Journal of Protein Chemistry》1998,17(3):273-278
Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed. 相似文献
16.
T Sano 《Bioconjugate chemistry》1999,10(5):905-911
A boron-enriched streptavidin has been prepared by chemical conjugation of a boron-rich compound, B(12)H(11)SH(2)(-) (BSH), to a genetically engineered streptavidin variant. The streptavidin variant used has 20 cysteine residues per molecule, derived from a C-terminal cysteine stretch consisting of five cysteine residues per subunit. Because natural streptavidin has no cysteine residues, the reactive sulfhydryl groups of the cysteine stretch serve as unique conjugation sites for sulfhydryl chemistry. BSH was conjugated irreversibly to the sulfhydryl groups of the streptavidin variant via a sulfhydryl-specific homobifunctional chemical cross-linker. Quantitative boron analysis indicates that the resulting streptavidin-BSH conjugate carries approximately 230 boron atoms/molecule. This indicates that the chemical conjugation of BSH to the streptavidin variant was highly specific and efficient because this method should allow the conjugation of a maximum of 240 boron atoms/streptavidin molecule. This boron-enriched streptavidin retained both full biotin-binding ability and tetrameric structure, suggesting that the conjugation of BSH has little, if any, effect on the fundamental properties of streptavidin. This boron-enriched streptavidin should be very useful as a component of targetable boron carriers for neutron capture therapy of cancer. For example, a monoclonal antibody against a tumor-associated antigen can be attached tightly to the boron-enriched streptavidin upon simple biotinylation, and the resulting conjugate could be used to target boron to tumor cells on which the tumor-associated antigen is overexpressed. 相似文献
17.
Norman Wang William F. Smith Brian R. Miller Dikran Aivazian Alexey A. Lugovskoy Mitchell E. Reff Scott M. Glaser Lisa J. Croner Stephen J. Demarest 《Proteins》2009,76(1):99-114
Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody VH and VL function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which VH and VL domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody VH and VL domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in VH and VL are at or adjacent to the VH–VL interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the VH domain, but not the VL domain, residue pairs at the variable‐constant domain interface (VH–CH1 interface) are also strongly conserved. The same network of conserved VH positions involved in interactions with both the VL and CH1 domains is found in camelid VHH domains, which have evolved to lack interactions with VL and CH1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc. 相似文献
18.
Background
Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet.Methodology/Principal Findings
We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. Vmax of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA.Conclusions/Significance
All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle. 相似文献19.
Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Potjanee Srimanote Kunan Bangphoomi Ornnuthchar Poungpair Santi Maneewatch Kiattawee Choowongkomon Wanpen Chaicumpa 《PloS one》2012,7(11)
NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/VHH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/VHH from a humanized-camel VH/VHH display library. VH/VHH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed VHH hallmark and were designated VHH6 and VHH24; other clones were conventional VH, designated VH9 and VH13. All VH/VHH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, VHH6 and VHH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/VHH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication. 相似文献
20.
BCL1 lymphoma protection induced by idiotype DNA vaccination is entirely dependent on anti-idiotypic antibodies 总被引:2,自引:0,他引:2
DNA vaccination with the idiotype (Id) of tumour B-cell membrane immunoglobulins (Ig) is a validated strategy to induce tumour protection to several mouse lymphomas. The relative contribution of anti-Id antibodies and T lymphocytes to tumour rejection is still debated. Previous studies in the BCL1 lymphoma model showed that scFv DNA immunisation induces a polyclonal antibody response restricted to conformational epitopes formed by the parental VL/VH association. We implemented a system based on this specificity to investigate the mechanism of BCL1 lymphoma protection induced by DNA immunisation. Antibody response and survival of mice immunised with the tumour Id scFv were compared with those of mice immunised simultaneously with two chimeric scFvs, containing either the tumour-derived VL or VH paired to an irrelevant VH or VL domain, respectively. Animals vaccinated with one or both chimeric constructs were not protected, despite the exposure to all putative tumour Id-derived MHC class I and class II T-cell epitopes. In addition, conformational antibodies induced by DNA vaccination caused tumour cells apoptosis and cell cycle arrest in vitro and transferred protection in vivo. Therefore, lymphoma rejection appears to be completely dependent on the induction of anti-Id antibodies. 相似文献