Thomas Metzler

Optimization of the Translation Initiation Region of Prokaryotic Expression Vectors: High Yield In Vitro Protein Expression and mRNA Folding

The process of in vitro protein synthesis that is based on T7 RNA polymerases differs strongly from what occurs in E. coli (Studier et al. 1990). Since the RNA polymerase of bacteriophage T7 works more than five times faster than endogenous RNA polymerases and the E. coli translation machinery, in vitro synthesized mRNAs are less protected by bacterial ribosomes. Consequently, no real coupling of prokaryotic transcription and translation can take place in vitro (Spirin 1999). As unprotected mRNAs easily form secondary structures, double-stranded regions can block the accessibility of important regulatory elements like the ribosomal binding site (RBS or Shine-Dalgarno site) and the start codon (AUG) and, thereby, inhibit the initiation of translation. Here, the initial region of prokaryotic expression vectors containing a T7 promoter and a T7 gene 10 enhancer was investigated and optimized for in vitro protein expression reactions.

Selective Labeling of Proteins in the RTS 500 System

The benefits of combining heteronuclear, multidimensional NMR with isotope-labeling strategies are well documented by recent studies of protein solution NMR spectroscopy. For example, the use of deuteration in concert with 13C-, 15N- labeling and the development of the TROSY technique have significantly increased the size of proteins that are amenable for study by NMR (Riek et al. 2000).