Tobacco Etch Virus Protease

Overview

Tobacco Etch Virus Protease (TEV protease) is a highly specific cysteine protease derived from Tobacco Etch Virus (TEV). With its remarkable sequence specificity, TEV protease has gained significant recognition as a valuable tool in the field of biochemistry and molecular biology. The catalytic domain of the nuclear inclusion protease from TEV is frequently used for cleaving genetically engineered fusion proteins.

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Introduction to TEV Protease

TEV possesses a unique characteristic in which its entire genetic material is encoded as a single massive polyprotein weighing approximately 350 kDa. To unleash the functional units encoded within this massive polyprotein, the virus relies on the actions of three distinct proteases: P1 protease, helper-component protease, and TEV protease. Each protease is responsible for cleaving the polyprotein at specific sites, with P1 protease and helper-component protease having one cleavage site each, while TEV protease has an impressive seven cleavage sites.

The TEV protease is a 27 kDa cysteine protease belonging to the 3C-type protease family. It plays a crucial role in processing the original polyprotein of TEV into functional viral proteins. Unlike serine proteases such as trypsin and chymotrypsin, TEV protease utilizes a thiol group from an active site cysteine residue for its catalytic activity. This unique characteristic ensures stringent sequence specificity, allowing the proper production of viral proteins. Due to its high specificity, TEV protease has gained popularity in the removal of affinity tags from recombinant proteins.

Structure of TEV Protease

The TEV protease belongs to the all β protein family. It has a distinctive two-domain structure with a β-barrel fold. In the first domain, the β sheet folds into an antiparallel β-barrel shape, while the β sheet in the second domain remains open. The β-barrel domain contains a characteristic Greek key motif. The catalytic triad, consisting of His46, Asp81, and Cys151, is located at the interface between the two domains.

Figure 1. Structure of TEV protease and the active site of TEV protease.
(Source: Nam, H. et al., 2021)

Structurally, TEV protease is most closely related to other 3C-type cysteine proteases found in the Picornaviridae virus family, including hepatitis A virus, poliovirus, foot and mouth disease virus, and rhinovirus. These proteases serve similar functions in their respective viruses. While their overall folds are similar, the specific atomic coordinates of TEV protease and these related proteins differ significantly. The root mean square deviation for α carbons between them ranges from 2.4 to 3.5 Å, indicating structural variations at the atomic level.

Specificity of TEV Protease

The preferred cleavage sequence for TEV protease was identified by analyzing recurring cut sites in the native polyprotein substrate. The consensus sequence for these cut sites is ENLYFQ\S, where '\' denotes the cleaved bond. Further studies characterized the protease's specificity by examining the cleavage of similar substrates. While ENLYFQ\S is the optimal sequence, the protease can cleave a range of substrates to varying degrees, indicating some substrate promiscuity. The protease shows the highest cleavage efficiency for sequences closest to the consensus EXLYΦQ\φ, where X represents any residue, Φ denotes a large or medium hydrophobe, and φ represents a small hydrophobic or polar residue.

Figure 2. Substrate binding sites of TEV protease.
(Source: Nam, H. et al., 2021)

The specificity of TEV protease is attributed to the extensive contact area between the enzyme and substrate. Unlike proteases such as trypsin, which possess shallow binding clefts with limited substrate interactions, TEV protease features a long C-terminal tail that completely covers the substrate, creating a binding tunnel. This tunnel contains tight binding pockets that complementarily accommodate each side chain of the substrate peptide, ensuring precise recognition and cleavage.

Applications of TEV Protease

TEV protease is widely utilized as a biochemical tool, particularly in the removal of affinity tags from purified recombinant fusion proteins. Its high sequence specificity enables controlled protein cleavage when the preferred sequence is inserted into flexible loops. This aspect makes TEV protease valuable in various research applications, including protein engineering, structural biology, and functional studies.

One of the notable advantages of TEV protease as a biochemical tool is its relatively low toxicity in vivo. The recognized cleavage sequence ENLYFQ\S is scarce in naturally occurring proteins, reducing the chances of unintended cleavage. Additionally, TEV protease has been subjected to directed evolution, allowing for modifications in its preferred residue positions before or after the cleavage site.

However, it is important to note that TEV protease does have certain limitations. It is prone to self-cleavage or autolysis, which can result in enzyme deactivation. This issue can be mitigated through a specific S219V mutation in the internal cleavage site. Furthermore, the solubility of TEV protease when expressed alone can be a challenge, although strategies such as fusion with maltose binding protein (MBP) have been implemented to enhance its solubility.

Reference

Nam H, et al. Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-leavage exhibits enhanced enzymatic activity. FEBS Open Bio. 2020, 10(4): 619-626.

Reagent Solutions

TEV Protease-Related Antibody

Target	Cat. No.	Product Name	Host	Isotype	Application
TEV Protease	DPAB22625	Anti-TEV Protease Polyclonal antibody	Rabbit	IgG	WB, ELISA	Inquiry
TEV P1 Protease	DPABH-29493	Anti-TEV P1 Protease Polyclonal antibody	Rabbit	IgG	WB	Inquiry
Trypsin	DCABH-8839	Anti-Trypsin monoclonal antibody, clone 21-215.2	Mouse	IgG2b	ELISA	Inquiry
	DCABH-8840	Anti-Trypsin monoclonal antibody, clone 22-324.3	Mouse	IgG2b	ELISA	Inquiry
	DCABH-8841	Anti-Trypsin monoclonal antibody, clone 22-347.8	Mouse	IgG2b	ELISA	Inquiry
	DCABH-5790	Anti-Trypsin monoclonal antibody, clone 3D5	Mouse	IgG1	sELISA, ELISA, WB	Inquiry
	DCABH-5779	Anti-Trypsin monoclonal antibody, clone 8D9	Mouse	IgG1	sELISA, WB, ELISA	Inquiry

TEV Protease-Related Antigen

Target	Cat. No.	Product Name	Expression System	Tag/Conjugate	Application
TEV Protease	DAGC574	Recombinant TEV Protease (S219V) [GST]	E. coli	GST	N/A	Inquiry
TEV Protease	DAG3632	Recombinant TEV Protease protein [His]	E. coli	His	N/A	Inquiry
EN-TEV Protease	DAG-P2272	EN-TEV Active EN-TEV Protease (aa 1 - 94)(mutation S219 N)	E. coli	Unconjugated	SDS-PAGE	Inquiry
TEV preM protein	DAG2398	Recombinant TEV PreM/M Protein (a.a. 2-168) [GST]	E. coli	GST	WB, ELISA	Inquiry
TEV Core protein	DAG1997	Recombinant TEV Core Protein (a.a. 1-96) [GST]	E. coli	GST	WB, ELISA	Inquiry
TEV N1a protein	DAG1876	Recombinant TEV N1a Protein [His]	E. coli	His	N/A	Inquiry

TEV Protease-Related ELISA

Target	Cat. No.	Product Name	Size	Species Reactivity	Application	Detection Sample
TEV	DEIAPV271	Tobacco etch virus (TEV) ELISA Kit	500T/1000T/5000T		Qualitative	host plants	Inquiry
Trypsin	DEIA-NS2310-14	Trypsin ELISA kit	96T	N/A	Quantitative	Biological samples	Inquiry
Chymotrypsin	DEIA10041	Human Chymotrypsin ELISA Kit	96T	Human	Quantitative	stool	Inquiry

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