Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13
| Autor: | Gunars Valkirs, William D. Husea, Diane McGrath, Grace R. Nakayama |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Phage display
Genes Viral biology Chemistry Phagemid Immunology Gene Expression Periplasmic space biology.organism_classification Bioinformatics Immunoglobulin light chain Bacteriophage Capsid Solubility Biochemistry Immunoglobulin Fragments Mutagenesis Site-Directed Cysteine Disulfides Site-directed mutagenesis Bacteriophage M13 |
| Zdroj: | Immunotechnology. 2:197-207 |
| ISSN: | 1380-2933 |
| DOI: | 10.1016/s1380-2933(96)00049-8 |
| Popis: | Background: Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage). Objectives: As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis. Study Design: One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage. Results: Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures. Conclusion: A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13. |
| Databáze: | OpenAIRE |
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