title: Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.
abstract: 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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St., 117997 Moscow, Russia. Electronic address: marine_gasparian@yahoo.com.
authors.forename: Marine E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Bobik
authors.forename: Yana V
authors.initials: YV
authors.lastname: Kim
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Ponomarenko
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Gabibov
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D061505:Cell Surface Display Techniques / Q000379:methods*; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D009281:Naphthalenes; D011993:Recombinant Fusion Proteins; C072587:2-naphthylamide; D004765:Enteropeptidase
keywords: Heterogeneous catalysis on phage surface; Phage display; Recombinant human enteropeptidase