MBP Squared

The Bioinformatics team have designed two PCR primers, one for each end of the Human MBP gene, that have different restriction enzyme cut sites and in one case, an octa-His motif added.  This run of eight Histidine residues will allow us to purify the MBP protein on a His-trap column.

The primers have been used to carry out the PCR amplification and the product was then cut with both Hind III and Eco RI to leave the different "sticky ends".  The host plasmid - pYES2 (a shuttle vector that can be used in  both E. coli and S. cerevisiae) was also cut with these two enzymes and the PCR product and plasmid were gel-purified and recovered.  Several ligation reactions were then set up to "stick" the PCR product into the pYES2 - the only way to know if this worked was to transform the re-constructed plasmid back into E. coli (generously donated by UoK), purify the DNA and then cut it with Eco RI and Hind III to release the human MBP gene.  This photo (taken on out new "blue light" transilluminator) shows that this has been successful.

The feint lower band is about 520 bp (the human gene) and the larger fragment is just under 6,000 bp and is compared to the cut vector in track 8.

We have sent one of the clones for sequencing and the results confrim that this is the human MBP gene.  The next step is to express the protein and confirm its identity by western blotting.