AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragme...AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system.METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS)at an interval of 3 wk. Group 1: PBS only; group 2:gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminalfragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 ℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination.RESULTS: The average titer of serum anti-HBsAg antibodyin the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay.CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.展开更多
The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative di...The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 （APLP2） C-terminal fragments （CTFs; C57, C50 and C31） in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein ex-pression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection in Alzheimer’s disease.展开更多
Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa...Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.展开更多
Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the meth...Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the methods to catalogue these macromolecules by detecting their interactions, modifications, and cellular locations. It will be helpful for scientists to compare the difference between a diseased cellular state and its normal state and to find the potential therapy treatment to intervene this status. One technology called split-protein reassembly or protein fragment complementation has been developed in the last two decades. This technology makes use of appropriate fragmentation of some protein reporters and the refolding of these reports could be detected by their function to confirm the interaction of interest. This system has been set up in cell-free systems, </span><i><span style="font-family:Verdana;">E.</span></i></span><i><span style="font-family:""> </span></i><i><span style="font-family:Verdana;">coli</span></i><span style="font-family:""><span style="font-family:Verdana;">, yeast, mammalian cells, plants and live animals. Herein, I present the development in fluorescence- and bioluminescence-based split-protein biosensors in both binary and ternary systems. In addition, some people developed the split-protein system by combining it with chemical inducer of dimerization strategy (CID). This has been applied for identifying the enzyme inhibitors and regulating the activity of protein kinases and phosphatases. With effort from many laboratories from the world, a variety of split-protein systems have been developed for studying the PPI </span><i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> and </span><i><span style="font-family:Verdana;">in vivo</span></i><span style="font-family:Verdana;">, monitoring the biological process, and controlling the activity of the enzyme of interest.展开更多
The deposition of amyloid-beta is a pathological hallmark of Alzheimer＇s disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase （beta-site amyloid pr...The deposition of amyloid-beta is a pathological hallmark of Alzheimer＇s disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase （beta-site amyloid precursor protein-cleaving enzyme 1） and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer＇s disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer＇s disease.展开更多
This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and...This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains.Myofibril fragmentation index,pH,the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured.These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles(P<0.05).The pH of psoas major muscle was the lowest at 0.5 h postmortem,and the highest after 12 h postmortem(P<0.05).In addition,the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles(P<0.05).The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently.This study gives a new perspective of the mechanism of postmortem meat tenderization.展开更多
A new phasing procedure has been proposed for dealing with single isomorphous replacement （SIR） x-ray diffraction data. The procedure combines SOLVE/RESOLVE with the dual-space fragment extension involving OASIS. Tw...A new phasing procedure has been proposed for dealing with single isomorphous replacement （SIR） x-ray diffraction data. The procedure combines SOLVE/RESOLVE with the dual-space fragment extension involving OASIS. Two sets of SIR data at 0.28 nm resolution taken from the protein （R）-phycoerythrin （PDB code： 1LIA） were used in the test. For one of the two SIR data sets, a default run of SOLVE/RESOLVE based on the heavy-atom substructure found by SHLEXD led automatically to an interpretable electron density map. OASIS could not effectively improve the result. For the other set of SIR data, SOLVE/RESOLVE resulted in a fragmented model consisting of 454 of the total 668 residues, in which only 29 residues were docked into the sequence. Based on this model, 7 iteration cycles of OASIS-DM- RESOLVE （build only） yielded automatically a model of 547 residues with 133 residues docked into the sequence. The overall-averaged phase error decreased considerably and the quality of electron density map was improved significantly. Two more cycles of iterative OASIS-DM-RESOLVE were carried out, in which the output phases and figures of merit from DM were merged with that from the original run of SOLVE/RESOLVE before they were passed onto RESOLVE （build only）. This led automatically to a model containing 452 residues with 173 docked into the sequence. The resultant electron density map is manually traceable. It is concluded that when results of SOLVE/RESOLVE are not sufficiently satisfactory, the combination of SOLVE/RESOLVE and OASIS-DM-RESOLVE （build only） may significantly improve them.展开更多
The program OASIS4.0 has been released. Apart from the improved single-wavelength anomalous diffraction （SAD） phasing algorithm described in a separate paper, an important new feature in this version is the automati...The program OASIS4.0 has been released. Apart from the improved single-wavelength anomalous diffraction （SAD） phasing algorithm described in a separate paper, an important new feature in this version is the automation of the iterative phasing and model-building process in solving protein structures. A new graphical user＇s interface （GUI） is provided for controlling and real-time monitoring the dual-space iterative process. The GUI is discussed in detail in the present paper.展开更多
Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired imm...Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacoki- netic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding;2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.展开更多
AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNA...AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.展开更多
基金Supported by the Major State Basic Research Development Program of China, Program 973, Grant No. 2001CB510001
文摘AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system.METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS)at an interval of 3 wk. Group 1: PBS only; group 2:gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminalfragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 ℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination.RESULTS: The average titer of serum anti-HBsAg antibodyin the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay.CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.
基金supported by the Natural Science Foundation of Technology Gallery in Jilin Province of China,No.2011-15237the National Natural Science Foundation of China,No.81160159
文摘The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 （APLP2） C-terminal fragments （CTFs; C57, C50 and C31） in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein ex-pression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection in Alzheimer’s disease.
基金supported by the National Natural Science Foundation of China,No.30360100,30760234,30860260,81160373,81360458
文摘Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
文摘Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the methods to catalogue these macromolecules by detecting their interactions, modifications, and cellular locations. It will be helpful for scientists to compare the difference between a diseased cellular state and its normal state and to find the potential therapy treatment to intervene this status. One technology called split-protein reassembly or protein fragment complementation has been developed in the last two decades. This technology makes use of appropriate fragmentation of some protein reporters and the refolding of these reports could be detected by their function to confirm the interaction of interest. This system has been set up in cell-free systems, </span><i><span style="font-family:Verdana;">E.</span></i></span><i><span style="font-family:""> </span></i><i><span style="font-family:Verdana;">coli</span></i><span style="font-family:""><span style="font-family:Verdana;">, yeast, mammalian cells, plants and live animals. Herein, I present the development in fluorescence- and bioluminescence-based split-protein biosensors in both binary and ternary systems. In addition, some people developed the split-protein system by combining it with chemical inducer of dimerization strategy (CID). This has been applied for identifying the enzyme inhibitors and regulating the activity of protein kinases and phosphatases. With effort from many laboratories from the world, a variety of split-protein systems have been developed for studying the PPI </span><i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> and </span><i><span style="font-family:Verdana;">in vivo</span></i><span style="font-family:Verdana;">, monitoring the biological process, and controlling the activity of the enzyme of interest.
基金supported by the National Natural Science Foundation of China, No. 81171192XMU Basic Training Program of Undergraduate, No. CXB2011019Visiting Scholar Fellowship of Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering of Xiamen University, No. 201101
文摘The deposition of amyloid-beta is a pathological hallmark of Alzheimer＇s disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase （beta-site amyloid precursor protein-cleaving enzyme 1） and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer＇s disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer＇s disease.
基金funded by the National Agricultural Science and Technology Innovation Program in China
文摘This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum,semitendinosus,and psoas major muscles during postmortem ageing for 5 d.These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains.Myofibril fragmentation index,pH,the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured.These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles(P<0.05).The pH of psoas major muscle was the lowest at 0.5 h postmortem,and the highest after 12 h postmortem(P<0.05).In addition,the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles(P<0.05).The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently.This study gives a new perspective of the mechanism of postmortem meat tenderization.
文摘A new phasing procedure has been proposed for dealing with single isomorphous replacement （SIR） x-ray diffraction data. The procedure combines SOLVE/RESOLVE with the dual-space fragment extension involving OASIS. Two sets of SIR data at 0.28 nm resolution taken from the protein （R）-phycoerythrin （PDB code： 1LIA） were used in the test. For one of the two SIR data sets, a default run of SOLVE/RESOLVE based on the heavy-atom substructure found by SHLEXD led automatically to an interpretable electron density map. OASIS could not effectively improve the result. For the other set of SIR data, SOLVE/RESOLVE resulted in a fragmented model consisting of 454 of the total 668 residues, in which only 29 residues were docked into the sequence. Based on this model, 7 iteration cycles of OASIS-DM- RESOLVE （build only） yielded automatically a model of 547 residues with 133 residues docked into the sequence. The overall-averaged phase error decreased considerably and the quality of electron density map was improved significantly. Two more cycles of iterative OASIS-DM-RESOLVE were carried out, in which the output phases and figures of merit from DM were merged with that from the original run of SOLVE/RESOLVE before they were passed onto RESOLVE （build only）. This led automatically to a model containing 452 residues with 173 docked into the sequence. The resultant electron density map is manually traceable. It is concluded that when results of SOLVE/RESOLVE are not sufficiently satisfactory, the combination of SOLVE/RESOLVE and OASIS-DM-RESOLVE （build only） may significantly improve them.
基金Project supported by the Innovation Foundation of the Chinese Academy of Sciences,and the National Basic Research Program of China(Grant No.2002CB713801)
文摘The program OASIS4.0 has been released. Apart from the improved single-wavelength anomalous diffraction （SAD） phasing algorithm described in a separate paper, an important new feature in this version is the automation of the iterative phasing and model-building process in solving protein structures. A new graphical user＇s interface （GUI） is provided for controlling and real-time monitoring the dual-space iterative process. The GUI is discussed in detail in the present paper.
文摘Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacoki- netic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding;2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.
文摘AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.
