Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens
Abstract
:1. Introduction
2. Results
2.1. #7TR Light Chain
2.1.1. Ni-NTA Column Chromatography (Route “1”)
2.1.2. Cation Exchange Chromatography (as a Second Step Purification)
2.1.3. Size-Exclusion Chromatography (as the Second Step Purification)
2.1.4. pH Dependency
2.1.5. Effect of Other Components (Reagents)
NaCl
Tween-20
2.1.6. Kinetics Using #7TR Purified by Route ”5” (pH 7.4)
2.2. H34 Light Chain
2.2.1. Purification (I) at pH 8.0 (Ni-NTA Chromatography)
2.2.2. Purification (II) at pH 5.5 (Cation-Exchange Chromatography)
2.2.3. Purification (III) at pH 7.4 (Size-Exclusion Chromatography)
2.2.4. Comparison of Ni-NTA (I) and Size-Exclusion (III) Chromatography
2.2.5. Kinetics
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Synthesis of FRET Substrates
4.3. Amplification of DNA Fragments Encoding Light Chains
4.4. Sequencing
4.5. Culture, Recovery, and Purification
4.6. Ni-NTA Column Chromatography (Route “1”)
4.7. Cation Exchange Chromatography (Route “2 & 3”)
4.7.1. Purification at pH 5.5 (Route “2”)
4.7.2. Purification at pH 8.0 (Route “3”)
4.8. Size-Exclusion Chromatography (Route “4–8”)
4.8.1. Purification at pH 5.5 (Route “4”)
4.8.2. Purification at pH 7.4 (Route “5”)
4.8.3. Purification at pH 8.0 (Route “6”)
4.8.4. Purification at pH 5.5 (Route “7”)
4.8.5. Purification at pH 8.0 (Route “8”)
4.9. Cleavage Assays
4.9.1. Arg(R)-pNA Substrate
4.9.2. FRET-Aβ and FRET-PD1 Substrates
4.10. Kinetics
4.10.1. Arg(R)-pNA
4.10.2. FRET-PD-1
5. Conclusions
- (1)
- The catalytic antibody should be prepared under a basic condition. In contrast, the catalytic activity is hindered or lost when prepared under an acidic condition. The appropriate pH range for the preparation was from 7.0 to 9.0. The low catalytic activity under the acidic pH is caused by the protonation of the aspartate participating in the catalytic site. Another possibility is not excluded, where the conformational structure of the catalytic site may be modulated during the progress of chromatography.
- (2)
- The presence of NaCl works to enhance or keep the high catalytic activity.
- (3)
- Surfactant of Tween-20 works to decrease the catalytic activity a little.
- (4)
- Size-exclusion chromatography is better than cation exchange chromatography.
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Nonaka, T.; Taguchi, H.; Uda, T.; Hifumi, E. Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens. Int. J. Mol. Sci. 2022, 23, 14351. https://doi.org/10.3390/ijms232214351
Nonaka T, Taguchi H, Uda T, Hifumi E. Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens. International Journal of Molecular Sciences. 2022; 23(22):14351. https://doi.org/10.3390/ijms232214351
Chicago/Turabian StyleNonaka, Tamami, Hiroaki Taguchi, Taizo Uda, and Emi Hifumi. 2022. "Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens" International Journal of Molecular Sciences 23, no. 22: 14351. https://doi.org/10.3390/ijms232214351