Frayne Consultants


Phosphate analogues that block nuclease acitivity in and out of cells. 
mRNA accumulator for the rapid increase in mRNA levels in bacteria
TLR9 agonist via modified single-stranded phage DNA
        

VivoSyn

  ┬«

Modulating Protein Synthesis

1) Shift the distribution of proteins in cells 

2) Increase total protein secretion in eukaryotic cells such as in yeast 2-4X fold 

3) Promotes differentiated state


Background: VivoSyn┬« modified phosphate can be used to globally shift the distribution of proteins in the cell impacting ~ one third of total cellular or secreted proteins. The average fold increase is 6 fold and the average fold decrease is 5 fold. Some proteins are elevated to much higher levels. This provides a powerful research tool to probe the impact of global alterations on cellular outputs such a protein secretion, cellular functions, synthesis of organic chemicals, etc. May be useful in fermentation processes using synthetic biology and hererologous gene expression. Appears to result in tighter control of gene expression. Not shown are similar results with human HEK tissue culture cells.

Enhanced Protein Secretion in Yeast

2D Polyacrylamide Difference Gel of Bacillus Secretome 








 







Total protein secretion by  S. cerevisiae (ln 1) is increased dramatically (lns 2-6) after growth of cells in the presence of the modified phosphate. Lns 1-6 contain respectively 0%, 10%, 15%, 20%, 30%, and 40%  modified phosphate to phosphate ratios (weight equivalent) in the media. Total protein secretion is enhanced 2-4 fold. Protein distribution shifts with increasing % modified phosphate as seen in lns 5-6 where total protein secretion is the same as lns 2-4 but distribution is dramatically altered. Enzyme assays indicate a recombinant alkaline phosphatase activity is increased with 20-100% substitution (optimal >75% substitution, reaching a 10 to 20 fold enhancement).  

Global shift in protein distribution

Overlay of 2D polyacrylamide gels using secreted proteins from B. subtilis treated with and without phosphate analogue. The green represents proteins which were enhanced in the modified gel and the magenta indicates those reduced. White appears where spots had similar intensity. TCA ppt proteins were separated by a pH 4-8 IEF gradient and then run on 10% polyacrylamide gels.