Cloning, sequencing, and expression of Trypanosoma brucei dihydrolipoamide dehydrogenase

A gene encoding dihydrolipoamide dehydrogenase was isolated from Trypanosoma brucei genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. The DNA sequence reveals that it encodes a protein of 478 amino acids (Mr 49935) highly similar to previously sequenced dihydrolipoamide dehydrogenases. The gene was ligated into pMEX8 and expressed in an Escherichia coli mutant that lacks dihydrolipoamide dehydrogenase. Expression resulted in the appearance of dihydrolipoamide dehydrogenase activity concurrent with the production of a protein of the expected Mr as determined by SDS/PAGE and Western blotting