2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity
| Format: | General Document |
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| building | INTELEK Repository |
| collection | Online Access |
| collectionurl | https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 |
| copyright | Copyright©PWB2025 |
| country | Malaysia |
| date | 2022-04-14 |
| format | General Document |
| id | 15494 |
| institution | UniSZA |
| internalnotes | Sila masukkan subject wajib Dissertations, Academic untuk semua tesis.. Terima kasih |
| originalfilename | CLONING AND EXPRESSION OF MUTANT-CARBOHYDRATE BINDING MODULE FAMILY 40 (CBM40) FROM Vibrio cholerae NON-O1 SIALIDASE TO ENHANCE ITS BINDING AFFINITY (MASTER_2022).pdf |
| person | Gogula Selvi A/P Asang |
| recordtype | oai_dc |
| resourceurl | https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15494 |
| sourcemedia | Server storage Scanned document |
| spelling | 15494 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15494 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 General Document Malaysia Library Staff (Top Management) Library Staff (Management) Library Staff (Support) Terengganu Faculty of Bio-resources & Food Industry English application/pdf 1.5 Server storage Scanned document Universiti Sultan Zainal Abidin UniSZA Private Access UNIVERSITI SULTAN ZAINAL ABIDIN SAMBox 2.3.4; modified using iTextSharp™ 5.5.10 ©2000-2016 iText Group NV (AGPL-version) 2022-04-14 171 CLONING AND EXPRESSION OF MUTANT-CARBOHYDRATE BINDING MODULE FAMILY 40 (CBM40) FROM Vibrio cholerae NON-O1 SIALIDASE TO ENHANCE ITS BINDING AFFINITY (MASTER_2022).pdf Gogula Selvi A/P Asang Copyright©PWB2025 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity Cloning Cloning Carbohydrate binding module family 40 (CBM40) is a non catalytic protein domain found in carbohydrate active enzyme (glycoside hydrolase) that recognizes sialic acid as binding substrate. Removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. Due to that, proteins with weak binding affinity often use site-directed mutagenesis approach to enhance protein affinity via an avidity effect. In this study, wild type CBM40 (WT-CBM40) was genetically modified using site-directed mutagenesis technique to form a stable mutant CBM40 (M-CBM40) followed by protein expression, purification and biochemical characterization studies. The recombinant clone WT-CBM40 was constructed using PCR amplification approach consist of CBM40 from Vibrio cholerae Non-O1 sialidase. WT-CBM40 was genetically modified using site-directed mutagenesis method by single amino acid mutation at threonine 200th (Thr200) to asparagine 200th (Asn200). Optimization of the proteins expression were performed at different concentrations of isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 0.5 - 1.5 mM and incubation temperatures (18°C and 25°C) respectively. For purification of both proteins, Ni-NTA affinity purification was performed. Purified CBM40 constructs (WT-CBM40 and M-CBM40) were tested for biochemical characterizations, including buffers, pHs, metal ions, chemical reagents and substrate specificity. In silico analysis was performed using UCSF Chimera software and Arpeggio in order to evaluate the impact of single mutagenesis on WT-CBM40 on substrate binding affinity. In this study, both WT-CBM40 and M-CBM40 have been successfully cloned in pET22b(+) in E. coli BL21 (DE3). M-CBM40 has been confirmed carry the mutation site via DNA sequencing approach. Both CBM40 constructs were successfully expressed in E. coli BL21 (DE3) strain 18˚C when induced with 1.5 mM IPTG where maximum expression was achieved at 16 hours after post-induction time. Both purified CBM40 constructs exhibited a single band with molecular weight of 24kDa. The biochemical characterizations of WT-CBM40 and M-CBM40 led to the identification of optimal buffer, pH, metal ions, chemical reagents and substrate specificity that improved their protein stability. The results gained by in silico analysis revealed that the affinity of M-CBM40 was higher than WT-CBM40 due to increases in hydrogen binding sites with sialic acid. From this study, M-CBM40 contructs shows greater binding affinity compared to WT-CBM40 due to increases in hydrogen-bonding, hydrophobic as well as polar interactions towards sialic acid. The optimum characterization conditions established would further lead to the discovery of its true potential in enhancing substrate binding affinity and protein carbohydrate recognition, which underpins its broad applications in protein engineering fields. Dissertations, Academic Sila masukkan subject wajib Dissertations, Academic untuk semua tesis.. Terima kasih Gene Expression Carbohydrate-Binding Modules Thesis |
| spellingShingle | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| state | Terengganu |
| subject | Cloning Dissertations, Academic |
| summary | Carbohydrate binding module family 40 (CBM40) is a non catalytic protein domain found in carbohydrate active enzyme (glycoside hydrolase) that recognizes sialic acid as binding substrate. Removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. Due to that, proteins with weak binding affinity often use site-directed mutagenesis approach to enhance protein affinity via an avidity effect. In this study, wild type CBM40 (WT-CBM40) was genetically modified using site-directed mutagenesis technique to form a stable mutant CBM40 (M-CBM40) followed by protein expression, purification and biochemical characterization studies. The recombinant clone WT-CBM40 was constructed using PCR amplification approach consist of CBM40 from Vibrio cholerae Non-O1 sialidase. WT-CBM40 was genetically modified using site-directed mutagenesis method by single amino acid mutation at threonine 200th (Thr200) to asparagine 200th (Asn200). Optimization of the proteins expression were performed at different concentrations of isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 0.5 - 1.5 mM and incubation temperatures (18°C and 25°C) respectively. For purification of both proteins, Ni-NTA affinity purification was performed. Purified CBM40 constructs (WT-CBM40 and M-CBM40) were tested for biochemical characterizations, including buffers, pHs, metal ions, chemical reagents and substrate specificity. In silico analysis was performed using UCSF Chimera software and Arpeggio in order to evaluate the impact of single mutagenesis on WT-CBM40 on substrate binding affinity. In this study, both WT-CBM40 and M-CBM40 have been successfully cloned in pET22b(+) in E. coli BL21 (DE3). M-CBM40 has been confirmed carry the mutation site via DNA sequencing approach. Both CBM40 constructs were successfully expressed in E. coli BL21 (DE3) strain 18˚C when induced with 1.5 mM IPTG where maximum expression was achieved at 16 hours after post-induction time. Both purified CBM40 constructs exhibited a single band with molecular weight of 24kDa. The biochemical characterizations of WT-CBM40 and M-CBM40 led to the identification of optimal buffer, pH, metal ions, chemical reagents and substrate specificity that improved their protein stability. The results gained by in silico analysis revealed that the affinity of M-CBM40 was higher than WT-CBM40 due to increases in hydrogen binding sites with sialic acid. From this study, M-CBM40 contructs shows greater binding affinity compared to WT-CBM40 due to increases in hydrogen-bonding, hydrophobic as well as polar interactions towards sialic acid. The optimum characterization conditions established would further lead to the discovery of its true potential in enhancing substrate binding affinity and protein carbohydrate recognition, which underpins its broad applications in protein engineering fields. |
| title | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| title_full | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| title_fullStr | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| title_full_unstemmed | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| title_short | 2022_Cloning and Expression of Mutant-Carbohydrate Binding Module Family 40 (CBM40) From Vibrio cholerae Non-O1 Sialidase to Enhance Its Binding Affinity |
| title_sort | 2022_cloning and expression of mutant-carbohydrate binding module family 40 (cbm40) from vibrio cholerae non-o1 sialidase to enhance its binding affinity |