Expression and Purification of Zinc Finger Antiviral Protein

Abstract

Ever since the discovery of viruses so many years ago there have been researchers of many different fields racing to find the perfect inhibitor. Any and all discoveries of viral resistance have been taken into a spotlight to discover the mechanisms. Zinc Finger Antiviral Protein (ZAP) is found in the kidney and liver cells of Rattus norvegicus; this protein makes these cells more resistant to viral infection. Gao et al concluded that ZAP facilitates inhibition of the antiviral gene expression, one of the major propagation steps (rather than inhibition of infection ZAP affects viral expression) by binding viral mRNA. Since then ZAP has been shown to increase resistance against Moloney Murine Leukemia virus, Sindbis virus, Ebola virus, and Marburg virus. Activity of ZAP containing four CCCH zinc fingers seems to be dependent on the integrity of the second and fourth CCCH zinc fingers. With HIV in mind, which is also a retrovirus, we have concluded that ZAP will have profound and influential implications.Our goal is to determine the structure of the zinc-binding domain of ZAP using Nuclear Magnetic Resonance. Zap was expressed as a fusion protein in E. coli with several different cleavage conditions and purified using Immobilized Metal Ion Affinity Chromatography (IMAC) were screened and found unsuccessful in that the cleaved ZAP was insoluble. However, we are currently working on the smaller ZAP proteins that only contain two zinc fingers; their constructs show promising results in terms of solubility after cleavage.