Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems

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• 作者：Takahiro Hohsaka ; Daisuke Kajihara ; Yuki Ashizuka ; Hiroshi Murakami ; and Masahiko Sisido
• 刊名：Journal of the American Chemical Society
• 出版年：1999
• 出版时间：January 13, 1999
• 年：1999
• 卷：121
• 期：1
• 页码：34 - 40
• 全文大小：163K
• 年卷期：v.121,no.1(January 13, 1999)
• ISSN：1520-5126

文摘

Efficiencies of the incorporation of various nonnatural amino acids carrying aromatic side groupsinto streptavidin were examined. The aromatic amino acids were linked to a mixed dinucleotide, pdCpA, andthe resulting aminoacyl pdCpAs were coupled with tRNACCCG(-CA) to afford chemically aminoacylatedtRNACCCG's. Mutant streptavidin mRNA containing a CGGG 4 base codon at the Tyr83 site was preparedand added to an Escherichia coli in vitro translation system with the aminoacyl tRNACCCG. The expression ofthe full-length mutant streptavidins was confirmed by a Western blot analysis, and their biotin binding activitywas examined by a dot blot analysis. The Western blot analysis indicated that the efficiencies of the incorporationwere higher for aromatic groups with straight configurations than those with widely expanded or bendconfigurations. The incorporation efficiencies were also examined in a rabbit reticulocyte lysate. In the lattersystem, the efficiencies were markedly improved for nonnatural amino acids with large side groups such aspyrene and anthraquinone.