Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.
The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.
Finzi
Andrés
A
Laboratoire de Rétrovirologie Humaine, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, CP 6128, Succursale Centre-ville, Montreal, Que., Canada H3C 3J7.
Cloutier
Jonathan
J
Cohen
Eric A
EA
D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015693:Gene Products, nef / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D006493:Heparin; D006639:Histidine; D008670:Metals; D011994:Recombinant Proteins / Q000096:biosynthesis
D015693:Gene Products, nef; D008670:Metals; D011994:Recombinant Proteins; D006639:Histidine; D006493:Heparin