Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties
| Autor: | Jeng-Jong Shieh, Peter O. Olins, Maire Helena Caparon, William F. Hood, Kumnan Paik, Ann M. Donnelly, Bauer Christopher S, John P. McKearn, Sarah R. Braford, Polazzi Joseph O, John W. Thomas, Jon A. Klover, Alan Michael Easton, David L. Zeng, Mark Allen Abrams, Joseph K. Welply, Barbara K. Klein |
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| Rok vydání: | 1999 |
| Předmět: |
chemistry.chemical_classification
Cancer Research Protein subunit Mutagenesis (molecular biology technique) Cell Biology Hematology Protein engineering Biology Protein Engineering medicine.disease_cause Amino acid Structure-Activity Relationship Amino Acid Substitution Biochemistry chemistry Mutagenesis Genetics medicine Humans Structure–activity relationship Interleukin-3 Molecular Biology Escherichia coli Interleukin 3 G alpha subunit |
| Zdroj: | Experimental Hematology. 27:1746-1756 |
| ISSN: | 0301-472X |
| DOI: | 10.1016/s0301-472x(99)00118-6 |
| Popis: | A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected. |
| Databáze: | OpenAIRE |
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