| Autor: |
Klein BK; G.D. Searle, Monsanto Co., St. Louis, MO, USA. barbara.k.klein@monsanto.com, Olins PO, Bauer SC, Caparon MH, Easton AM, Braford SR, Abrams MA, Klover JA, Paik K, Thomas JW, Hood WF, Shieh JJ, Polazzi JO, Donnelly AM, Zeng DL, Welply JK, McKearn JP |
| Jazyk: |
angličtina |
| Zdroj: |
Experimental hematology [Exp Hematol] 1999 Dec; Vol. 27 (12), pp. 1746-56. |
| DOI: |
10.1016/s0301-472x(99)00118-6 |
| Abstrakt: |
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: |
MEDLINE |
| Externí odkaz: |
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