A COMPREHENSIVE ANALYSIS OF THE TOBACCO ETCH VIRUS PROTEASE P1' AMINO ACID SPECIFICITY

Abstract

Affinity tags have become indispensable tools for protein expression and purification. Not only can they increase the yield and stability of recombinant proteins, but in certain cases they can also enhance their solubility. However, because affinity tags have the potential to interfere with structural and functional studies, they must usually be removed at some point during the purification. Many of the proteases that are used to remove affinity tags do not have stringent specificity for their recognition sites, and this often results in the degradation of the target protein. On the other hand, tobacco etch virus (TEV) protease, which recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-▼-Gly/Ser (processing occurring as indicated after the glutamine residue), is highly specific for its target site; there have not been any reports of it cleaving at noncanonical sites. One potential limitation of TEV protease, however, is that it is believed to require a glycine or serine in the P1' position (the site immediately after the scissile bond) to be able to efficiently cleave its substrates, yet some proteins need amino acids other than glycine or serine at their N-terminus for them to be functionally active. To investigate the stringency of the requirement for a glycine or serine residue in the P1' position of the TEV protease recognition site, 20 variants of a fusion protein substrate with an otherwise optimum recognition site were constructed, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Only proline in the P1' position of the recognition site was completely inhibitory to processing and many of the P1' variants were readily cleaved by the protease. Consequently, these results demonstrate that TEV protease can tolerate a large variety of amino acids in the P1' position.