engleski

Suppressor tRNA mediated bacterial expression system for Acetabularia (Dasycladales, Chlorophyta) genes containing UAA and UAG glutamine codons.

The unicellular marine green alga Acetabularia uses a nonstandard genetic code, in which the universal stop codons UAA and UAG encode glutamine. Functional analysis of recombinant Acetabularia proteins would therefore be limited to analysis of partially translated polypeptides. To circumvent the problem of protein truncation, we introduced an inducible suppressor-tRNA into expression plasmid pET-5a. The UAA tRNA-suppressor inserts glutamic acid for codons UAA and UAG ; codon UAG is read by wobble pairing. Monitoring the expression of Acetabularia actin by Western blotting proved our system functional, showing that each of the three UAA and UAG codons in Acetabularia actin were partially successfully read through. Additionally, expressed polypeptides, whether truncated or not, can be used for epitope mapping experiments targeting interactive molecules as nucleic acids, proteins or other compounds.

Escherichia coli

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