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Generation of Anti-c-Met Single Domain Antibody Fragment Based on Human Stable Frameworks
Selvakumar Edwardraja,Sriram Sokalingam,Govindan Raghunathan,Bum-Yeol Hwang,이선구 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.6
The size reduction is an important issue in the biomedical application of antibody and single domain antibody fragment is recognized as very attractive tool. However, it is very time-consuming and laborious to generate the fragment antibody with targeted binding function. Here,we investigated the possibility to prepare single domain antibody (sdAb) by a simple grafting method based on stable human consensus framework sequences. The complementarity determining region sequences in VH domain of anti-c-Met scFv from rabbit were grafted with the human VH3 consensus framework sequences, which generated the anti-c-Met single domain antibody showing almost same binding activity to its scFv form. The generated single domain antibody could be produced as functional form in oxidizing cytoplasm of E. coli, but produced as inactive form in reducing cytoplasm. The structural analysis of the homology models gave us the insight on the stability of the single domain antibody. In this report, we have demonstrated that the very stable human consensus framework sequence can be used for the generation of active anti-c-Met sdAb via complementarity determining regions grafting. We expect that this kind of grafting method for the generation of sdAb may provide us with the opportunities to prepare sdAbs based on the known antibody sequences.
Selvakumar, Edwardraja,Rameshkumar, Neelamegam,Lee, Sun-Gu,Lee, Soo-Jae,Park, Hyung-Soon WILEY-VCH Verlag 2010 Chembiochem Vol.11 No.4
<B>Graphic Abstract</B> <P>Just where you want it: Grafting target scFv complementarity-determining regions onto stable frameworks (FR) and consensus-based engineering of methionines on the FR enables the production of methionine-free scFv that can be specifically modified with a bio-orthogonal reactive group on its N-terminal through in vivo incorporation of a methionine analogue. <img src='wiley_img_2010/14394227-2010-11-4-CBIC200900685-content.gif' alt='wiley_img_2010/14394227-2010-11-4-CBIC200900685-content'> </P>
Edwardraja, Selvakumar,Neelamegam, Rameshkumar,Ramadoss, Vijayaraj,Venkatesan, Subramanian,Lee, Sun-Gu Wiley Subscription Services, Inc., A Wiley Company 2010 Biotechnology and bioengineering Vol.106 No.3
<P>Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.</P>
Selvakumar Edwardraja,Ganapathiraman Munussami,Amit Goyal,이선구 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.6
There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs.
Ganapathiraman Munussami,Sriram Sokalingam,Selvakumar Edwardraja,김중래,정성욱,이선구 한국화학공학회 2018 Korean Journal of Chemical Engineering Vol.35 No.3
Cystatins and lipocalins have attracted considerable interest for their potential applications in non-immunoglobulin protein scaffold engineering. In the present study, their potential homologs were screened computationally from non-redundant protein sequence database based on the overlapped conserved residues (OCR)-fingerprints, which can detect the protein family with low sequence identity, such as cystatins and lipocalins. Two types of OCR-fingerprints for each family were designed and showed very high detection efficiency (>90%). The protein sequence database was scanned by the fingerprints, which yielded the hypothetical sequences for cystatins and lipocalins. The hypothetical sequences were validated further based on their sequence motifs and structural models, which allowed an identification of the potential homologs of cystatins and lipocalins.