An encoded N-terminal extension results in low levels of heterologous protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Samantha?S?Orchard Email author" target="_blank">Heidi?Goodrich-BlairEmail author |
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Institution: | (1) Department of Bacteriology, University of Wisconsin, 53706 Madison, WI, USA;(2) Department of Biology, San Diego State University, 92182 San Diego, CA, USA |
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Abstract: | Background The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid
sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start
codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form"
(using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in
an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium. |
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