Abstract
Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene of PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a(+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a(+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a(+)-PPDK]-expressed recombinant PPDK fused to an N-terminal sequence of 6-His tag. The molecular weight of PPDK was estimated to be 101 kDa by SDS-PAGE. The electrophoretic grade PPDK was obtained by His•Bind resin affinity chromatography and ultrafiltration using 10-kDa molecular weight cutoff membrane. The successful application of PPDK in pyrosequencing was also demonstrated.
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Zou, B., Song, Q., Zhou, G. (2016). Expression of PPDK from Microbispora rosea subsp. aerata in E. coli and Its Application in Pyrosequencing. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_18
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DOI: https://doi.org/10.1007/978-1-4939-3308-2_18
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