Simultaneous and site-directed incorporation of an ester linkage and an azide group into a polypeptide by in vitro translation.
A method is presented by which an azide-containing side chain can be introduced into any internal position of a polypeptide chain by in vitro translation. For this, 2'-deoxy-cytidylyl-(3'-->5')-adenosine was acylated on the 3'(2')-hydroxyl group of adenosine with 6-azido-2(S)-hydroxyhexanoic acid (AHHA), an alpha-hydroxy- and epsilon-azide derivative of L-lysine. The acylated dinucleotide was enzymatically ligated with a tRNA transcript to provide chemically stable E. coli suppressor AHHA-tRNA(Cys(CUA)). The esterase 2 gene from Alicyclobacillus acidocaldarius was modified by the amber stop codon (UAG) on position 118. Using AHHA-tRNA(Cys(CUA)) in an E. coli in vitro translation/transcription system, the site-directed introduction of an azide group linked to a backbone ester into the esterase polypeptide was achieved. The yield of the synthesized modified protein reached 80% compared to translation of the native esterase. Subsequently, azide coupling with an alkyne-modified oligodeoxynucleotide demonstrated the feasibility of this approach for conjugation of polypeptides.
Humenik
Martin
M
Laboratorium für Biochemie, Universität Bayreuth, Universitätstrasse 30, 95440 Bayreuth, Germany.
Huang
Yiwei
Y
Safronov
Igor
I
Sprinzl
Mathias
M
D000215:Acylation; D001386:Azides / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D004952:Esters / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D001386:Azides; D018388:Codon, Terminator; D004952:Esters; D010455:Peptides; D012343:RNA, Transfer; D004950:Esterases; D008239:Lysine