Abstract
A dATP conversion E. coli strain could be constructed when both pyk and adk1 gene were expressed successfully. pyk gene encodes pyruvate kinase (PK) could be expressed, when inserted it before adk1 gene which encodes adenylate kinase (AK) in plasmid pET-pyk-adk1 after transform into E. coli and the recombinant could be used to convert dATP from dAMP. Another plasmid pET-adk1-pyk, which inserted pyk gene behind of adk1, the recombinant E. coli transformed with this plasmid could not convert dAMP into dATP, pyk gene cannot be translated in this recombinant. The different translation levels of pyk with gene location switching caused mainly by the different secondary structures formed by the 5’-untranslation regions and the gene sequence of its5’-terminal. The dATP product E. coli strain could be constructed when cloned pyk gene at an optimum location.
Highlights
A Deoxyadenosine triphosphate (dATP) conversion E. coli strain could be constructed when both pyk and adk1 gene were expressed successfully. pyk gene encodes pyruvate kinase (PK) could be expressed, when inserted it before adk1 gene which encodes adenylate kinase (AK) in plasmid pET-pyk-adk1 after transform into E. coli and the recombinant could be used to convert dATP from dAMP. Another plasmid pET-adk1-pyk, which inserted pyk gene behind of adk1, the recombinant E. coli transformed with this plasmid could not convert dAMP into dATP, pyk gene cannot be translated in this recombinant
Deoxyadenosine triphosphate is one of the necessary precursors used in DNA biosynthesis, especially in the PCR reaction
Though there have two kinds of arranging castle for pyk gene and adk1 gene, only the recombinant E. coli BL21 could be used to catalyze 500 mM of dAMP converting to dATP
Summary
Deoxyadenosine triphosphate (dATP) is one of the necessary precursors used in DNA biosynthesis, especially in the PCR reaction. The commercial dATP products come mainly from chemical synthesis, but the chemical method gives relatively low yield and need the toxic substances issued by the Environmental Protection Agency, like pyridine and dimethylformamide is cited from [1]. The biosynthesis method is environmental friendly one, a couple phosphorylation reactions can be used to synthesis dATP for dAMP. In order to simplify the biosynthesis process, two kinases were cloned onto a high copy plasmid pET17b and performed overexpression in E. coli BL21. The recombinants are used to catalyze dATP from dAMP after inducting protein expression of plasmid pET-pykadk. The poor expression spectrum of pyk gene on the pET-adk1-pyk plasmid prevented the conversion of dAMP to dATP, and leading to the accumulation of intermediary dADP produced
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