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1.
001-es BibID:
BIBFORM040661
Első szerző:
Kapust, Rachel B.
Cím:
The P1' specificity of tobacco etch virus protease / Kapust Rachel B., Tözsér József, Copeland Terry D., Waugh David S.
Dátum:
2002
ISSN:
0006-291X
Megjegyzések:
Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, 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. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
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Megjelenés:
Biochemical And Biophysical Research Communications. - 294 : 5 (2002), p. 949-955. -
További szerzők:
Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Copeland, Terry D.
Waugh, David S.
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DOI
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2.
001-es BibID:
BIBFORM040662
Első szerző:
Kapust, Rachel B.
Cím:
Tobacco etch virus protease : mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency / Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., Waugh, D. S.
Dátum:
2001
ISSN:
1741-0126
Megjegyzések:
Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.
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Megjelenés:
Protein Engineering Design & Selection. - 14 : 12 (2001), p. 993-1000. -
További szerzők:
Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Fox, Jeffrey D.
Anderson, D. Eric
Cherry, Scott
Copeland, Terry D.
Waugh, David S.
Internet cím:
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DOI
Intézményi repozitóriumban (DEA) tárolt változat
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3.
001-es BibID:
BIBFORM040675
Első szerző:
Nallamsetty, Sreedevi
Cím:
Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro / Nallamsetty Sreedevi, Kapust Rachel B., Tözsér József, Cherry Scott, Tropea Joseph E., Copeland Terry D., Waugh David S.
Dátum:
2004
ISSN:
1046-5928
Megjegyzések:
Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4 degrees C. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease.
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Megjelenés:
Protein Expression And Purification. - 38 : 1 (2004), p. 108-115. -
További szerzők:
Kapust, Rachel B.
Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Cherry, Scott
Tropea, Joseph E.
Copeland, Terry D.
Waugh, David S.
Internet cím:
Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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