Campylobacter jejuni is a pathogenic bacterium which utilizes nitrate reduction for growth via a periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, is a molybdenum-containing 4Fe-4S protein. Heterologous expression of NapA has been carried out using a plasmid containing the napA gene with a 6x Histidine-tag and a second plasmid containing the napLD genes. However, the presence of a 6x His-tag in the protein can be problematic. Kinetic studies of the recombinant protein reveal a low affinity for nitrate (2.2 mM), compared to native Sulfurospirillum barnesii NapA (0.29 mM). Herein, molecular cloning, overexpression, and purification of napA without the His-tag were investigated. The expression plasmid, pET21-a(+), was cut with an alternative restriction enzyme, Bam-HI, to prevent His-tag incorporation. NapA was expressed in LB medium hosted in E.coli T7 strain induced with IPTG. After purification, enzyme kinetics was done to calculate the difference in activity relative to the His-tag purified protein.

Alternate molecular cloning and heterologous expression of NapA from <i>Campylobacter Jejuni</i>

243rd National Meeting (2012)

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Alternate molecular cloning and heterologous expression of NapA from Campylobacter Jejuni