Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points
| Autor: | Mehdi Arbabi-Ghahroudi, N. Gaudette, Tomoko Hirama, Rebecca To, Jamshid Tanha, Wen Ding, R. MacKenzie |
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| Rok vydání: | 2008 |
| Předmět: |
Protein Denaturation
Phage display Stereochemistry Immunoglobulin Variable Region Bioengineering Complementarity determining region Biochemistry Peptide Library Animals Humans Disulfides Isoelectric Point Peptide library Molecular Biology chemistry.chemical_classification Chi-Square Distribution Heavy-chain antibody biology Temperature Hydrogen-Ion Concentration Complementarity Determining Regions Molecular biology Isoelectric point Enzyme chemistry biology.protein Immunoglobulin heavy chain Directed Molecular Evolution Immunoglobulin Heavy Chains Camelids New World Systematic evolution of ligands by exponential enrichment Plasmids Biotechnology |
| Zdroj: | Protein Engineering Design and Selection. 22:59-66 |
| ISSN: | 1741-0134 1741-0126 |
| Popis: | When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s. |
| Databáze: | OpenAIRE |
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