Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 singie-chain Fv fragment （scFv） to the human IgG1 CH2 （i.e., Cγ2） and CH3 （i.e., Cγ3） domain...Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 singie-chain Fv fragment （scFv） to the human IgG1 CH2 （i.e., Cγ2） and CH3 （i.e., Cγ3） domains with the human IgG1 hinge （i.e. Hγ）. Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 （VH-Linker-VL-Hγ-Cγ2-Cγ3） remained its activity because of its preserved conformation, while the binding activity of the LH23 （VL-Linker-VH-Hγ-Cγ2-Cγ3） was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20^＋ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.展开更多
The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG ...The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant domain. Some similarity was found. These results suggest that the binding mechanism of protein G to the IgG Fc part depends on hydrophobic action which comes from the α-helix in protein G molecule, just as protein A binding to IgG Fc part.展开更多
The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA...The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA）, a cell-wall protein of Staphylococcus aureus, has been developed as a universal ligand for immunoglobulin G （IgG） purification because it binds specifically to the Fc portion of the IgG molecule of many mammals[2]. However, certain characteristics of SPA severely restrict the advancement of the antibody industry.展开更多
文摘Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 singie-chain Fv fragment （scFv） to the human IgG1 CH2 （i.e., Cγ2） and CH3 （i.e., Cγ3） domains with the human IgG1 hinge （i.e. Hγ）. Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 （VH-Linker-VL-Hγ-Cγ2-Cγ3） remained its activity because of its preserved conformation, while the binding activity of the LH23 （VL-Linker-VH-Hγ-Cγ2-Cγ3） was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20^＋ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.
文摘The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant domain. Some similarity was found. These results suggest that the binding mechanism of protein G to the IgG Fc part depends on hydrophobic action which comes from the α-helix in protein G molecule, just as protein A binding to IgG Fc part.
基金supported by grants from the National High Technology Research and Development Program of China(863 Program)(No.2012AA020304)the National Natural Science Foundation of China(Grant No.31300643)the Innovation Fund for Postgraduate Students from the Simcere Pharmaceutical Company(No.02704051)
文摘The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA）, a cell-wall protein of Staphylococcus aureus, has been developed as a universal ligand for immunoglobulin G （IgG） purification because it binds specifically to the Fc portion of the IgG molecule of many mammals[2]. However, certain characteristics of SPA severely restrict the advancement of the antibody industry.
