MOLECULAR-CLONING, SEQUENCE DETERMINATION AND HETEROLOGOUS EXPRESSIONOF NUCLEOSIDE DIPHOSPHATE KINASE FROM PISUM-SATIVUM

Protein sequence data derived from the N-terminal region of a 17 kDa p olypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were use d to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein enco ded by NDK-P1 had a calculated molecular mass of 16485 Da and possesse d substantial homology with nucleoside diphosphate kinases (NDKs) isol ated and cloned from other sources. High levels of expression of NDK-P 1 protein were achieved in Escherichia coli using a T7-driven expressi on system. Recombinant NDK-P1 protein was shown to possess NDK activit y and had similar biochemical characteristics to NDKs isolated from ot her sources. The Michaelis constants for a variety of nucleoside dipho sphate (NDP) substrates were found to be broadly similar to those repo rted for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.