LARGE SCALE PRODUCTION, PURIFICATION, AND 65CU SOLID STATE NMR OF AZURIN

Journal Article · Tue Jan 01 04:00:00 EST 2008 · Journal of Undergraduate Research

OSTI ID:1052092

Gao, A; Heck, R W

This paper details a way to produce azurin with an effi ciency over 10 times greater than previously described and demonstrates the fi rst solid state nuclear magnetic resonance spectrum of 65Cu(I) in a metalloprotein. A synthetic gene for azurin based upon the DNA sequence from Pseudomonas aeruginosa including the periplasmic targeting sequence was subcloned into a T7 overexpression vector to create the plasmid pGS-azurin, which was transformed into BL21 (DE3) competent cells. The leader sequence on the expressed protein causes it to be exported to the periplasmic space of Escherichia coli. Bacteria grown in a fermentation unit were induced to overexpress the azurin, which was subsequently purifi ed through an endosmotic shock procedure followed by high performance liquid chromatography (HPLC). 1,500 mg of azurin were purifi ed per liter of culture. 65Cu(II) was added to apo-azurin and then reduced. The 65Cu metal cofactor in azurin was observed with solid state nuclear magnetic resonance (NMR) to determine any structural variations that accompanied copper reduction. This is the fi rst solid state NMR spectra of a copper(I) metalloprotein. Analysis of the NMR spectra is being used to complement hypotheses set forth by x-ray diffraction and computational calculations of electron transfer mechanisms in azurin.

• Research Organization: DOESC (USDOE Office of Science (SC) (United States))
• Sponsoring Organization: USDOE Office of Science (SC)
• OSTI ID: 1052092
• Journal Information: Journal of Undergraduate Research, Journal Name: Journal of Undergraduate Research Vol. 8
• Country of Publication: United States
• Language: English