Expression of human thymidylate synthase in Escherichia coli [unknown]

V J Davisson, W Sirawaraporn, D V Santi
1994 Journal of Biological Chemistry  
We recently discovered that the recombinant human thymidylate synthase (TS) described contains a mixture of authentic human TS plus a fusion protein with 13 amino acids added to the N terminus. We have now made a construct that eliminates the fusion. Purified enzyme from this construct has a specific activity of 0.6 unitslmg, which is indistinguishable from that reported. Expression was originally obtained in a vector that utilized a 115-base pair 5'-untranslated leader sequence from the
more » ... nce from the Lactobacillus casei TS gene. This leader sequence contains potential start codons at -13 and -32 codons upstream from the authentic start codon (Fig. I) . We had previously documented that only the authentic start site was used in the expression of L. casei TS in Escherichia coli, but we were unable to directly sequence the N terminus of human TS at the time of the above report. Recently we performed mass spectral analysis of the molecular weight of human TS and found that the major component of the preparation had a subunit molecular weight of 37,074 c -7 7 8, considerably greater than the predicted weight of 35,717 (or 35,585 without the N-terminal methionine). N-terminal sequence analysis revealed a major sequence of -RVVNG (-80%) and a minor sequence of PVAGSE (-10-20%). The minor sequence corresponds to the authentic human TS sequence, while the major sequence corresponds to a translational start at the -13 Met codon (see Fig. 1 ). The -13 start is in accord with the measured molecular weight. Examination of SDS gels of several preparations indicates variation in the utilization of the authentic and upstream starts. We inserted a stop codon at the -2 codon (Fig. 1) using polymerase chain reaction mutagenesis. Human TS was purified from extracts of cells containing the new construct, pGCHTS-TAA. This construct expresses human TS at about the same level as the original one. Extract from 2 liters of culture yielded -5 mg of pure enzyme with a specific activity of 0.6 units/mg. SDS-polyacrylamide gel electrophoresis of this enzyme showed a single band. The molecular weight determined by mass spectrometry is 35,588 2 6, in perfect agreement with the predicted molecular weight of 35,585 without the N-terminal methionine.
pmid:7982996 fatcat:ggzyxfe34jczdbxlxq7qhds5am