کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1952161 1057182 2013 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase
کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase
چکیده انگلیسی


• Active human enteropeptidase catalytic chain (L-HEP) was expressed on M13 phage.
• Heterogeneous catalysis by phage-bound L-HEP was shown on peptide substrate.
• Kinetic parameters were similar to those of enzyme in solution.
• Fusion proteins containing a D4K cleavage site were also cleaved.

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s−1. Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volume 95, Issue 11, November 2013, Pages 2076–2081
نویسندگان
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