Cloning,sequence and expression in Escherichia coli of the gene encoding phosphofructokinase from Bacillus macquariensis |
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| Institution: | 1. Department of Botany, University of Wisconsin-Madison, Madison, WI, United States;2. Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan;1. Department of Information Engineering, via G. Gradenigo, 6b, University of Padova, 35131 Padova, Italy;2. Department of Biomedical Sciences, University of Padova, via U. Bassi, 58, 35131 Padova, Italy;3. ARC – Centro Ricerche Applicate s.r.l., via J. Da Montagnana, 49, 35132 Padova, Italy;1. Department of Chemistry and Biochemistry, California State University Los Angeles, Los Angeles, CA 90032, USA;2. Department of Chemistry and Biochemistry, California State University Northridge, CA 91330, USA |
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| Abstract: | A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable. |
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