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001-es BibID:	BIBFORM120789
035-os BibID:	(Scopus)85191248548 (WoS)001208395200001
Első szerző:	Golda Mária (molekuláris biológus)
Cím:	P1' specificity of the S219V/R203G mutant tobacco etch virus protease / Mária Golda, Gyula Hoffka, Scott Cherry, Joseph E. Tropea, George T. Lountos, David S. Waugh, Alexander Wlodawer, József Tőzsér, János András Mótyán
Dátum:	2024
ISSN:	0887-3585
Megjegyzések:	Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk tobacco etch virus protein structure molecular dynamics fusion tag removal enzymology TEV protease protease Proteolysis
Megjelenés:	Proteins-Structure Function And Bioinformatics. - 92 : 9 (2024), p. 1085-1096. -
További szerzők:	Hoffka Gyula (1992-) (vegyész) Cherry, Scott Tropea, Joseph E. Lountos, George T. Waugh, David S. Wlodawer, Alexander Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Mótyán János András (1981-) (biokémikus, molekuláris biológus)
Pályázati támogatás:	TKP2021-EGA-20 Egyéb ÚNKP-23-5-DE-486 Egyéb BO/00110/23/5 MTA 75N91019D00024 Egyéb
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001-es BibID:	BIBFORM040670
Első szerző:	Mahalingam, Bhuvaneshwari
Cím:	Combining mutations in HIV-1 protease to understand mechanisms of resistance / Mahalingam Bhuvaneshwari, Boross Peter, Wang Yuan-Fang, Louis John M., Fischer Christopher C., Tozser Jozsef, Harrison Robert W., Weber Irene T.
Dátum:	2002
ISSN:	0887-3585
Megjegyzések:	HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Proteins-Structure Function And Bioinformatics. - 48 : 1 (2002), p. 107-116. -
További szerzők:	Boross Péter (1972-) (biokémikus, vegyész) Wang, Yuan-Fang Louis, John M. Fischer, Christopher C. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Harrison, Robert W. Weber, Irene T.
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001-es BibID:	BIBFORM126434
Első szerző:	Mótyán János András (biokémikus, molekuláris biológus)
Cím:	Characterization of the E26H Mutant Schistosoma japonicum Glutathione S-Transferase / Mótyán János András, Nagyné Veres Ágota, Tőzsér József
Dátum:	2025
ISSN:	0887-3585
Megjegyzések:	Glutathione-S-transferase, such as that of Schistosoma japonicum (sjGST) belongs to the most widely utilized fusion tags in the recombinant protein technology. The E26H mutation of sjGST has already been found to remarkably improve its ability for binding divalent ions, enabling its purification with immobilized metal affinity chromatography (IMAC). Nevertheless, most characteristics of this mutant remained unexplored to date. In this study, we performed a comparative analysis of the wild-type and the E26H mutant sjGST by using in vitro as well as in silico approaches. We confirmed that the sjGST(E26H) protein exhibits significantly increased affinity for binding nickel ions as compared to the wild-type. In addition, we proved that the sjGST(E26H) can be purified efficiently either with glutathione- or immobilized metal ion-affinity chromatography, even in consecutive purification steps. The human retroviral-like aspartic protease 1 (ASPRV1) conjugated with the sjGST(E26H) fusion tag was also successfully purified by using both of these affinity chromatographic approaches. Our studies revealed that the E26H mutant sjGST can be used as a versatile affinity tag because the modified protein retains the kinetic features of the wild-type and its affinity towards glutathione, while can be purified efficiently by IMAC, as well.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Schistosoma japonicum recombinant protein protein purification GST mutagenesis glutathione S-transferase fusion tag affinity chromatography
Megjelenés:	Proteins-Structure Function And Bioinformatics. - 93 : 5 (2025), p. 1054-1066. -
További szerzők:	Veres Ágota (laboráns) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:	TKP2021-EGA-20 Egyéb ÚNKP-22-2-I-DE-391 Egyéb ÚNKP-23-5-DE-486 Egyéb BO/00110/23/5 MTA University of Debrecen (UD) Program for Scientific Publication Egyéb UD Faculty of Medicine Research Fund (Bridging fund) Egyéb UD Scientific Research Bridging Fund (DETKA) Egyéb
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001-es BibID:	BIBFORM001504
Első szerző:	Tie, Yunfeng
Cím:	Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir / Tie Y., Kovalevsky A. Y., Boross P., Wang Y. F., Ghosh A. K., Tozser J., Harrison R. W., Weber I. T.
Dátum:	2007
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Proteins 67 : 1 (2007), p. 232-242. -
További szerzők:	Kovalevsky, Andrey Yu Boross Péter (1972-) (biokémikus, vegyész) Wang, Yuan-Fang Ghosh, Arun K. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Harrison, Robert W. Weber, Irene T.
Internet cím:	elektronikus változat DOI
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