C-terminal modifications of a protein by UAG-encoded incorporation of puromycin during in vitro protein synthesis in the absence of release factor 1

被引:12
作者
Agafonov, DE [1 ]
Rabe, KS [1 ]
Grote, M [1 ]
Voertler, CS [1 ]
Sprinzl, M [1 ]
机构
[1] Univ Bayreuth, Biochem Lab, D-95440 Bayreuth, Germany
关键词
fluorescent probes; protein engineering; protein modifications; puromycin; translation;
D O I
10.1002/cbic.200500358
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80% yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed However, the conjugation yield decreased upon addition of more than six nucleotides.
引用
收藏
页码:330 / 336
页数:7
相关论文
共 26 条
[1]   Efficient suppression of the amber codon in E-coli in vitro translation system [J].
Agafonov, DE ;
Huang, YW ;
Grote, M ;
Sprinzl, M .
FEBS LETTERS, 2005, 579 (10) :2156-2160
[2]   The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis [J].
Agafonov, DE ;
Rabe, KS ;
Grote, M ;
Huang, YW ;
Sprinzl, M .
FEBS LETTERS, 2005, 579 (10) :2082-2086
[3]   THE 3'-END OF TRANSFER-RNA AND ITS ROLE IN PROTEIN-BIOSYNTHESIS [J].
CHLADEK, S ;
SPRINZL, M .
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 1985, 24 (05) :371-391
[4]  
CHLADEK S, 1985, ANGEW CHEM, V97, P377
[5]   An active role for tRNA in decoding beyond codon:anticodon pairing [J].
Cochella, L ;
Green, R .
SCIENCE, 2005, 308 (5725) :1178-1180
[6]   GENERATION OF A HYBRID SEQUENCE-SPECIFIC SINGLE-STRANDED DEOXYRIBONUCLEASE [J].
COREY, DR ;
SCHULTZ, PG .
SCIENCE, 1987, 238 (4832) :1401-1403
[7]   Peptide ligation and its application to protein engineering [J].
Cotton, GJ ;
Muir, TW .
CHEMISTRY & BIOLOGY, 1999, 6 (09) :R247-R256
[8]   A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation [J].
Dinçbas-Renqvist, V ;
Engström, Å ;
Mora, L ;
Heurgué-Hamard, V ;
Buckingham, R ;
Ehrenberg, M .
EMBO JOURNAL, 2000, 19 (24) :6900-6907
[9]   Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions [J].
Doi, N ;
Takashima, H ;
Kinjo, M ;
Sakata, K ;
Kawahashi, Y ;
Oishi, Y ;
Oyama, R ;
Miyamoto-Sato, E ;
Sawasaki, T ;
Endo, Y ;
Yanagawa, H .
GENOME RESEARCH, 2002, 12 (03) :487-492
[10]   Peptide conjugates as tools for the study of biological signal transduction [J].
Eisele, F ;
Owen, DJ ;
Waldmann, H .
BIOORGANIC & MEDICINAL CHEMISTRY, 1999, 7 (02) :193-224