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Volumn 7, Issue 2, 2006, Pages 249-252

Termination-free prokaryotic protein translation by using anticodon-adjusted E. coli tRNASer as unified suppressors of the UAA/UGA/UAG stop codons. Read-through ribosome display of full-length DHFR with translated UTR as a buried spacer arm

Author keywords

Biotechnology; Nonsense suppression; Proteins; Proteomics; Ribosome display

Indexed keywords

ADENINE; DIHYDROFOLATE REDUCTASE; GUANINE; PROTEIN; TRANSFER RNA; URACIL;

EID: 32344435560     PISSN: 14394227     EISSN: None     Source Type: Journal    
DOI: 10.1002/cbic.200500397     Document Type: Article
Times cited : (8)

References (26)
  • 5
    • 13444265467 scopus 로고    scopus 로고
    • Angew. Chem. Int. Ed. 2005, 44, 1048-1053.
    • (2005) Angew. Chem. Int. Ed. , vol.44 , pp. 1048-1053
  • 6
    • 0024968835 scopus 로고
    • Nonsense suppression is a naturally occurring phenomenon and has been widely used to site-specifically incorporate an unnatural amino acid or analogue into proteins by using an anticodon-adjusted and chemically misacylated suppressor tRNA. Examples are: a) C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188;
    • (1989) Science , vol.244 , pp. 182-188
    • Noren, C.J.1    Anthony-Cahill, S.J.2    Griffith, M.C.3    Schultz, P.G.4
  • 10
    • 32344450002 scopus 로고    scopus 로고
    • note
    • Determined by the band length of the DHFR ORF-UTR domain amplified by PCR from the total cDNA of the E. coli MG1655 strain by using a forward primer specific to the 5′-terminal of the DHFR ORF. Total cDNA was prepared from total mRNA by using a BD SMART mRNA Amplification Kit (BD Biosciences, USA) in combination with Poly(A) Tailing Kit (Ambion, USA).
  • 12
    • 32344442610 scopus 로고    scopus 로고
    • note
    • There is a small but notable difference in the band intensities for the RT-PCR amplified T7(+)(78) and T7(-)(78) templates.
  • 13
    • 4644254373 scopus 로고    scopus 로고
    • The corresponding yields are ≤0.2% (in the presence of anti-ssrA oligonucleotide and protein disulfide isomerase) in the conventional ribosome display by using stop-codon-deleted mRNAs that have a sufficiently long (> 100 aa) 3′ or C-terminal arm. The remarkably enhanced yields obtained here are most likely due to the use of reconstituted translation systems, which are much less contaminated with RNases and other unfavorable components, such as ssrA RNA, and thus protect the mRNA templates from degradation. See, for example: a) A. C. Forster, V. W. Cornish, S. C. Blacklow, Anal. Biochem. 2004, 333, 358-364;
    • (2004) Anal. Biochem. , vol.333 , pp. 358-364
    • Forster, A.C.1    Cornish, V.W.2    Blacklow, S.C.3
  • 23
    • 32344452153 scopus 로고    scopus 로고
    • note
    • Ser, see ref. [13b].
  • 24
    • 32344448224 scopus 로고    scopus 로고
    • note
    • Only the T7(+) template was recovered from a 1:1 mixture of T7(+) and T7(-) templates under competitive conditions (Figure S1 in the Supporting Information).
  • 25
    • 32344453224 scopus 로고    scopus 로고
    • note
    • Ser in read-through of each (ochre, opal, or amber) template (Figure 3c and d) leaves little doubt that Ser is introduced at each stop codon.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.