Inhibition of tobacco etch virus protease activity by detergents Arun K. Mohanty, Chad R. Simmons, and Michael C. Wiener * Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736, USA Received 3 June 2002, and in revised form 22 August 2002 Abstract Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein–detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and fre- quently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, a 1 platelet ac- tivating factor acetylhydrolase (PAFAHa 1 ). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins. Ó 2002 Elsevier Science (USA). All rights reserved. Biochemical and structural characterization of inte- gral membrane proteins require multimilligram quanti- ties of purified protein. Recombinant DNA technology has facilitated the design and development of various vector DNA based systems for high-level expression of foreign genes in different hosts. For the purification of recombinant proteins, various classical separation pro- cedures are employed and are based on the physical- chemical properties of the protein, such as charge, size, and hydrophobicity. These procedures are often time consuming and laborious; therefore, simple and rapid alternatives for purification have been developed. The production of recombinant proteins containing poly- histidine tags has become a frequently used procedure for rapid purification of recombinant proteins [1,2]. This affinity tagging is readily performed by fusion of a cDNA that codes for polyhistidine (typically six to ten histidines) to the gene of interest in the expression vec- tor, which yields a protein that can be detected and/or purified via immobilized metal affinity chromatography (IMAC) or anti-histidine antibodies. Purification of many bacterially expressed integral membrane proteins has been reported (for example, glucose transporter [3], diacylglycerol kinase [4], outer membrane iron sidero- phore transporter FhuA [5], nontypeable Haemophilus influenzae p5 outer membrane protein [6], CLC chloride channel homolog YadQ [7], and acyl–acyl carrier pro- tein synthase [8]). Removal of the affinity tag from the protein of interest is frequently sought and is accom- plished by inserting a site-specific protease cleavage site between the tag and the protein. The tag is then sepa- rated from the fusion protein by proteolysis after affinity chromatography. Many different proteases are used for cleavage including Factor Xa [9], thrombin [10,11], en- terokinase [12], preScission [13], or tobacco etch virus Protein Expression and Purification 27 (2003) 109–114 www.elsevier.com/locate/yprep * Corresponding author. Fax: +1-434-982-1616. E-mail address: mwiener@virginia.edu (M.C. Wiener). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-5928(02)00589-2