Pseudolysin (PLN) protein is a major virulent factor in Pseudomonas aeruginosa infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by Streptomyces nigrescens. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by P. aeruginosa. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography and gel filtration. His tag was removed by digestion with tobacco etch virus (TEV) protease. Proteolytic activity of tagged or untagged PLN, monitored as the rate of release of p-nitroanilide (pNa) from N-succinyl-ala-ala-ala-p-nitroanilide (N-SucAla3-pNa) at 410 nm, was assayed in the presence and absence of SMPI. Analysis of data showed that in the absence of SMPI, untagged PLN was two-fold more active (46 pmolpNa/sec) to hydrolyze N-SucAla3-pNa compared to His-tagged PLN (23 pmolpNa/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmolpNa/sec while that of untagged PLN remained the same. Doubling the concentration of SMPI (ratio 1:2 PLN to SMPI) reduced the enzymatic activity of untagged PLN by 50 % (23 pmol pNa/sec). The outcomes of this study implied that His tag at the N terminus of PLN impacted its enzymatic activity and its interaction with SMPI.
","abstract_html":"<p>Pseudolysin (PLN) protein is a major virulent factor in <em>Pseudomonas</em> <em>aeruginosa </em>infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by <em>Streptomyces nigrescens</em>. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by <em>P</em>. <em>aeruginosa</em>. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography and gel filtration. His tag was removed by digestion with tobacco etch virus (TEV) protease. Proteolytic activity of tagged or untagged PLN, monitored as the rate of release of <em>p</em>-nitroanilide (<em>p</em>Na) from <em>N</em>-succinyl-ala-ala-ala-<em>p</em>-nitroanilide (<em>N</em>-SucAla<sub>3</sub>-<em>p</em>Na) at 410 nm, was assayed in the presence and absence of SMPI. Analysis of data showed that in the absence of SMPI, untagged PLN was two-fold more active (46 pmol<em>p</em>Na/sec) to hydrolyze <em>N</em>-SucAla<sub>3</sub>-<em>p</em>Na compared to His-tagged PLN (23 pmol<em>p</em>Na/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmol<em>p</em>Na/sec while that of untagged PLN remained the same. Doubling the concentration of SMPI (ratio 1:2 PLN to SMPI) reduced the enzymatic activity of untagged PLN by 50 % (23 pmol <em>p</em>Na/sec). The outcomes of this study implied that His tag at the N terminus of PLN impacted its enzymatic activity and its interaction with SMPI.</p>","abstract_has_math":false,"creators":["Adewole, Adewale Adeboye"],"institution":null,"degree_name":"Master of Science in Natural Science","degree_level":"Thesis","degree_discipline":"Chemistry and Biochemistry","degree_department":null,"school":null,"contributors":["Odutayo Odunuga, Ph.D.","Bidisha Sengupta, Ph.D.","Darrell Fry, Ph.D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-08-08T07:00:00Z","date_published":"2025-08-08T07:00:00Z","updated_at":"2026-07-24T04:30:49Z","subjects":["Pseudolysin","Drug Development","Pseduomonas aeruginosa","Streptomyces metalloprotease inhibitors","antimicrobial resistance","Protein interactions","enzymatic assay","Biochemistry","Molecular Biology","Pathogenic Microbiology","Pharmaceutical Preparations","Pharmacology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://scholarworks.sfasu.edu/etds/626","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Odutayo Odunuga, Ph.D.","Bidisha Sengupta, Ph.D.","Darrell Fry, Ph.D."]},{"key":"dc:creator","label":"Author","values":["Adewole, Adewale Adeboye"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2025-08-06T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry and Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science in Natural Science"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Pseudolysin","Drug Development","Pseduomonas aeruginosa","Streptomyces metalloprotease inhibitors","antimicrobial resistance","Protein interactions","enzymatic assay","Biochemistry","Molecular Biology","Pathogenic Microbiology","Pharmaceutical Preparations","Pharmacology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarworks.sfasu.edu/etds/626"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Pseudolysin (PLN) protein is a major virulent factor in Pseudomonas aeruginosa infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by Streptomyces nigrescens. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by P. aeruginosa. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography and gel filtration. His tag was removed by digestion with tobacco etch virus (TEV) protease. Proteolytic activity of tagged or untagged PLN, monitored as the rate of release of p-nitroanilide (pNa) from N-succinyl-ala-ala-ala-p-nitroanilide (N-SucAla3-pNa) at 410 nm, was assayed in the presence and absence of SMPI. Analysis of data showed that in the absence of SMPI, untagged PLN was two-fold more active (46 pmolpNa/sec) to hydrolyze N-SucAla3-pNa compared to His-tagged PLN (23 pmolpNa/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmolpNa/sec while that of untagged PLN remained the same. Doubling the concentration of SMPI (ratio 1:2 PLN to SMPI) reduced the enzymatic activity of untagged PLN by 50 % (23 pmol pNa/sec). The outcomes of this study implied that His tag at the N terminus of PLN impacted its enzymatic activity and its interaction with SMPI.
"]},{"key":"dc:title","label":"Title","values":["EFFECT OF HISTIDINE TAG ON THE ENZYMATIC ACTIVITY OF PSEUDOLYSIN AND ITS INTERACTION WITH THE STREPTOMYCES METALLOPROTEASE INHIBITOR (SMPI) PROTEIN"]}]}],"canonical_facts":{"dc:contributor":["Odutayo Odunuga, Ph.D.","Bidisha Sengupta, Ph.D.","Darrell Fry, Ph.D."],"dc:creator":["Adewole, Adewale Adeboye"],"dc:date.available":["2025-08-06T07:00:00Z"],"dc:description.abstract":["Pseudolysin (PLN) protein is a major virulent factor in Pseudomonas aeruginosa infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by Streptomyces nigrescens. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by P. aeruginosa. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography and gel filtration. His tag was removed by digestion with tobacco etch virus (TEV) protease. Proteolytic activity of tagged or untagged PLN, monitored as the rate of release of p-nitroanilide (pNa) from N-succinyl-ala-ala-ala-p-nitroanilide (N-SucAla3-pNa) at 410 nm, was assayed in the presence and absence of SMPI. Analysis of data showed that in the absence of SMPI, untagged PLN was two-fold more active (46 pmolpNa/sec) to hydrolyze N-SucAla3-pNa compared to His-tagged PLN (23 pmolpNa/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmolpNa/sec while that of untagged PLN remained the same. Doubling the concentration of SMPI (ratio 1:2 PLN to SMPI) reduced the enzymatic activity of untagged PLN by 50 % (23 pmol pNa/sec). The outcomes of this study implied that His tag at the N terminus of PLN impacted its enzymatic activity and its interaction with SMPI.
"],"dc:identifier":["https://scholarworks.sfasu.edu/etds/626"],"dc:subject":["Pseudolysin","Drug Development","Pseduomonas aeruginosa","Streptomyces metalloprotease inhibitors","antimicrobial resistance","Protein interactions","enzymatic assay","Biochemistry","Molecular Biology","Pathogenic Microbiology","Pharmaceutical Preparations","Pharmacology"],"dc:title":["EFFECT OF HISTIDINE TAG ON THE ENZYMATIC ACTIVITY OF PSEUDOLYSIN AND ITS INTERACTION WITH THE STREPTOMYCES METALLOPROTEASE INHIBITOR (SMPI) PROTEIN"],"thesis:degree_discipline":["Chemistry and Biochemistry"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science in Natural Science"]},"updated_at":"2026-07-24T04:30:49Z"}