Content area
Abstract
The cell-free protein synthesis (CFPS) system is a versatile method for protein production. This approach uses an Escherichia coli based, in vitro combined transcription-translation system for producing proteins from a DNA template. By optimizing reagents, modifying host strains used for cell extract, and improving methods for processing, the cellular components are used effectively.
We investigate the efficiency of CFPS to incorporate unnatural amino acids into the proteins while maintaining high yields. We utilize native tRNA aminoacyl synthetases to acylate a tRNA with selenomethionine, selenocysteine, or fluorophenylalanine; amino acids useful for protein structural studies. When the native amino acid is replaced by the amino acid analog in CFPS reactions, the mutant protein yields are indistinguishable from wild-type protein yields.
Incorporation efficiency was determined by purifying proteins using metal chelate affinity and ion exchange chromatography. Comparing intact molecular weight of the wild-type and mutant protein using mass spectrometry determines the extent of unnatural amino acid incorporation. Furthermore, using protein digests coupled with reverse-phase HPLC and tandem mass spectrometry, we determined the replacement efficiency at specific residues. The mass spectrum identifies peaks, and quantification is determined from the peak areas of the HPLC UV absorbance. Selenomethionine was completely incorporated in our system. Selenocysteine incorporation was investigated as well as the dual incorporation of selenomethionine and selenocysteine.
Fluorophenylalanine showed variable incorporation efficiency. To further improve the efficiency of incorporation, an auxotrophic strain unable to synthesize phenylalanine was constructed. This strain along with changes in the cell-extract preparation procedure resulted in improved incorporation efficiency without a significant loss in protein yield. Higher fluorophenylalanine concentrations and the use of extracts from the auxotrophic strain both result in improved incorporation efficiency.
In summary, my research utilized various methods for investigating in vitro protein production. The use of various molecular biology tools has been essential to this investigation and to the improvement of the CFPS platform. Additional techniques allow quantification of various proteins relevant to biological reactions. In particular, the coupling of mass spectrometry with HPLC-UV spectra has allowed for improved assessment of unnatural amino acid incorporation.
