Covalent immobilization of tobacco‐etch‐virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins

Abstract

Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97%) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 degrees C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

“…Despite TEVp's popularity as an efficient reagent for removal of fusion tags from recombinant target proteins [19], [20] and in biomedical research [21], [22], [23], thorough characterization of its substrate specificity by applying combinatorial library approaches have until recently been lacking [24]. Instead, our knowledge of the substrate specificity has largely been based on alignment and comparison of naturally occurring processing sites of TEVp as well as cleavage analysis of substrate variants representing a relatively limited set of amino acid replacements at relevant positions within the TEVp substrate consensus sequence, ENLYFQG [25], [26].…”

Sharka: The Past, The Present and The Future

“…In addition, NIa proteinase possesses structural motifs shared with cellular serine proteases with the substitution of serine by a cysteine as the active site nucleophile. The specificity of enzyme and control its activity is discussed in some recently published papers [72,73,74], which are focused on the investigation of recombinant viruses and their host interactions. Mutation in the Lys(27) of NIa-Pro has established the role of this enzyme in the determination of host specificity.…”

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

“…Consequently, a lot of efforts around the world are dedicated to the characterization of proteases and their substrates (2,31). In addition to their biological importance, proteases have attracted much interest in several biotechnological and industrial applications, such as removal of "fusion tags" from recombinant target proteins (38), as supplements in dishwashing and laundry detergents, or for bating of hides and skin in the leather industry (41,44). Sometimes, however, their use is hindered due to limitations inherent to a specific protease: for example, low solubility, poor enzyme stability and specificity, or limited activity.…”