I got several comments on/off board for this. I have checked by Western blotting if any expression was occurring. I did not see any expression in either the inclusion body or soluble fraction.
It appears that I have solved the problem; Several papers suggest that the 10-30 bases after the start codon affect protein expression due to the secondary structure of the mRNA. Comparison of the 30 bases after the start codon of the entire protein family using the Vienna RNAfold server (http://rna.tbi.univie.ac.at/) shows a more negative free energy of the thermodynamic ensemble for the proteins which I can not express compared to the ones I can. I have taken the DNA sequence of one that does not express and converted it to mRNA. I used the mRNA optimiser program (http://bioinformatics.ua.pt/software/mrna-optimiser/) to maximizing the molecule minimum free energy resulting in decreased secondary structure strength. This was converted back to DNA and the gene synthesized. I now see expression of the protein (still in the inclusion body). I don't know exactly why it now works as several parameters were changed during mRNA optimization (changes in codons, GC content and 2nd structure of mRNA). I thought I would share in case anyone else wanted to try. Thanks and enjoy your weekend! Dan
