with 70 ethanol the dna was dissolved in sterile distilled water and digested with ecori the dna fragments were electrophoretically fractionated in a 0 7 low melting agarose gel upon ethidium bromide staining and ultraviolet illumination a unique 2600 bp band was revealed this band was sliced from the gel and the agarose was melted at 70 u00b0 c the dna was then phenol extracted and precipitated with alcohol the 2600 bp ecori fragment was then recloned using standard procedures at the ecori site of the plasmid expression vector ptrchis c obtained from invitrogen this vector has the same reading frame as u03bbgt11 contains all the dna sequences to obtain high level protein expression in e coli and also contains a sequence encoding 6 consecutive histidine residues which allow the expressed protein to bind ni charged 34 probond 34 resin invitrogen so that the recombinant protein can be easily purified in a one step procedure the ptrchis c plasmid containing the 2600 bp fragment was transformed into e coli strain top10 obtained from invitrogen expression of the recombinant protein was demonstrated by western blot analysis transformants were grown in luria broth lb at 37 u00b0 c to an od 600 of 0 5 then isopropylthio u03b2 d galactoside iptg a gratuitous inducer of the lac operon was added to a final concentration of 1 mm to induce expression of the recombinant protein the transformants were grown an additional 3 hours at 30 u00b0 c after induction then about 200 u03bcl of culture was placed in a microfuge tube and centrifuged.