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Research in Pharmaceutical Sciences، جلد ۱۸، شماره ۵، صفحات ۵۴۱-۵۵۰
عنوان فارسی
چکیده فارسی مقاله
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عنوان انگلیسی A novel approach to enhance the performance of kallikrein 6 enzyme using Pichia pastoris GS115 as a host
چکیده انگلیسی مقاله Background and purpose: Enzyme engineering is the process of raising enzyme efficiency and activity by altering amino acid sequences. Kallikrein 6 (KLK6) enzyme is a secreted serine protease involved in a variety of physiological and pathological activities. The increased expression of KLK6 plays a key role in various diseases. Instability and spontaneous activation and deactivation are major challenges in the study of this enzyme. This study aimed to create a stable pro-KLK6 enzyme by enzyme engineering, designing a specific cleavage site for enterokinase, and using Pichia pastoris GS115 as a host cell. Then, recombinant pro-KLK6 was used to introduce a novel inhibitor for it. Experimental approach: An engineered pro-KLK6 gene was cloned into the pPICZα A expression vector. Then, it was expressed in P. pastoris GS115 and purified by Ni-NTA chromatography. An inactive engineered pro-KLK6 gene was cleaved by enterokinase and converted to an active KLK6. The KLK6 enzyme activity and its kinetic parameters were measured using N-benzoyl-L-arginine ethyl ester (BAEE) substrates. Findings/Results: The secretory form of the pro-KLK6 was expressed at about 11 mg/L in P. pastoris (GS115). Before activation with enterokinase, pro-KLK6 was inactive and did not activate spontaneously. The kinetic parameters, including K m and V max, were estimated at 113.59 µM and 0.432 μM/s, respectively. Conclusion and implications: A stable pro-KLK6 enzyme was produced using P. pastoris (GS115) as the host cell and a specific cleavage site for enterokinase. Additionally, this study assessed the kinetic parameters of the KLK6 enzyme using the BAEE substrate for the first time.
کلیدواژه‌های انگلیسی مقاله Enterokinase,Kallikrein6,Kinetic,Nα-Benzoyl-L-arginine ethyl ester,P. pastoris (GS115).
نویسندگان مقاله | Fatemeh Mahmoodi
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.


| Hamid Bakherad
Biophysics and Computational Biology Laboratory (BCBL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.


| Navid Mogharrab
Department of Pharmacology and Toxicology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.


| Mohammad Rabbani


نشانی اینترنتی http://rps.mui.ac.ir/index.php/jrps/article/view/2211
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زبان مقاله منتشر شده en
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نوع مقاله منتشر شده Original Article
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