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Volumn 288, Issue 5473, 2000, Pages 2042-2045

Designing small-molecule switches for protein-protein interactions

Author keywords

[No Author keywords available]

Indexed keywords

HUMAN GROWTH HORMONE; INDOLE DERIVATIVE;

EID: 0034674624     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5473.2042     Document Type: Article
Times cited : (95)

References (31)
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    • note
    • 2+-nitrilotriacetic acid column.
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    • Structures of the compounds are available on Science Online
    • Structures of the compounds are available on Science Online at www.sciencemag.org/feature/data/1050853. shl or upon request from the authors.
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    • 104 → Gly-hGHbp (1.5 mg/ml), 150 (μl of binding buffer [1 × phosphate-buffered saline (PBS) (pH 7.4) and 1 mM EDTA], and 2 μl of a 10 mM stock solution of ligand in DMSO. The mixture was then incubated at room temperature for 30 min and centrifuged at 18,000g for 10 min. The supernatant was subsequently incubated at room temperature for 30 min with 50 μl of Streptavidin Magnetic Particles (Boehringer Mannheim), which were preblocked with bovine serum albumin (BSA). The captured phages were washed (5 × I ml) with binding buffer supplemented with 0.1% Tween 20, 0.1% BSA, and 1% of 10 mM ligand stock solution in DMSO. Finally, the phage particle suspension (10 μl out of 100 μl in binding buffer) was used directly to superinfect 1 ml of midlogarithmic XL1-Blue culture (absorbance at 600 nm = 0.5 to 1.0) and titered on LB ampicillin (100 μg/ml) agar plates.
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    • 175 → Gly mutant.
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    • a is the association constant.
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    • 201 → Cys-hGHbp) portions were then fused through PCR reaction to obtain the full-length receptor gene. The gene was inserted into modified pEGFP-N1 (Clontech), whose replication origin was replaced by the low copy number replication origin from plasmid pACYC177 (New England Biolabs) between the Xho I and Not I sites. The resulting mutant receptor gene sequence was confirmed. However, the engi-neered stop codon TGA was removed in this construct, and the extra sequence AAATLDHNQPYHICRGFTCFKKPPTPPPEPET was added. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E. Glu; F, Phe; G, Gly H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; T, Thr, and Y, Tyr.
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    • note
    • ueous Non-Radioactive Cell Proliferation Assay (Promega) and normalized to IL-3 response at 5 units/ml.
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    • note
    • 175 → Gly-hGH in the FDC-P1 cell proliferation assay.
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    • The authors acknowledge funding from the Norvartis Research Foundation
    • The authors acknowledge funding from the Norvartis Research Foundation.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.