ScienceAsia 29 (2003): 347-353 |doi: 10.2306/scienceasia1513-1874.2003.29.347

Cloning and Effective Induction of Escherichia coli Nucleoside Diphosphate Kinase by Lactose

Pattama Howhan^a and Somchai Pornbanlualap^a,b,*

ABSTRACT: The complete PCR-derived DNA fragment containing the structural gene for Escherichia coli nucleoside diphosphate kinase (ndk) gene was cloned into pET-26b, a T₇ promoter-based expression vector. After transforming recombinant pET-ndk into the E. coli BL21(DE3) pLysS, the expression of ndk gene was induced by addition of either IPTG or lactose. Surprisingly, lactose was found to be as effective inducer as IPTG. Both inducers gave almost the same level of expression of ndk gene. The rate of induction of nucleoside diphosphate (NDP) kinase by lactose, however, occurred at a much slower rate than by IPTG. Because the pET system had become one of the most powerful and commonly used vector for expressing recombinant proteins, this study to our knowledge is the first to demonstrate that lactose can be substituted for the more costly IPTG as inducer. Furthermore, because the kinetics of induction by lactose occurred at a much slower rate than IPTG, lactose may be the preferred inducer over IPTG in the situation where induction of recombinant protein resulted in formation of insoluble inclusion bodies. The recombinant NDP kinase containing polyhistidine tagged at the C-terminus was purified in a single step by Ni²⁺-NTA affinity chromatography. The purified enzyme contained two major bands, one that migrated at 16 KDa which corresponding to monomeric form and the other at 32 KDa which corresponding to the dimeric form of enzyme. This purification protocol resulted in a yield of 132 mg of protein per one liter of cell culture. The purified recombinant enzyme that contained polyhistidine tagged at the C-terminus retained its activity, with the specific activity of 2,620 units/mg. This polyhistidine tagged NDP kinase can be reversibly immobilized onto Ni²⁺-NTA agarose in a column for a more efficient, semi-solid state synthesis of deoxyribonucleoside 5'- triphsophate.

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a Interdisciplinary Graduate Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok, Thailand
b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand

* Corresponding author, Email: fsciscpl@ku.ac.th

Received 4 Mar 2003, Accepted 14 Aug 2003