title: Strategy for improvement of enteropeptidase efficiency in tag removal processes.
abstract: Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. It has also great biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occurred during cleavage of fusions when high amount of enzyme is required. In this study we have improved the efficiency of fusion proteins cleavage by enteropeptidase by substitution of the Lys residue by Arg in specific cleavage sequence (Asp)(4)-Lys. We have demonstrated that 3-6-fold lower amounts of the catalytic subunit of human and bovine enteropeptidase is required for 95% cleavage of Trx/TRAIL and Trx/FGF-2 fusions with (Asp)(4)-Arg cleavage sequence in comparison to native sequence (Asp)(4)-Lys. As a result, reduced amount of non-specifically cleaved peptide fragments were observed during cleavage of (Asp)(4)-Lys/Arg mutated fusions. These findings overcome limitations of enteropeptidase in tag removal processes during recombinant proteins purification and extend its commercial benefit in the biopharmaceutical industry.
authors.affiliation: Department of Bioengineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997 Moscow, Russia. marine_gasparian@yahoo.com
authors.forename: Marine E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: Maxim L
authors.initials: ML
authors.lastname: Bychkov
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D014362:Trypsinogen / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D016222:Fibroblast Growth Factor 2; D013879:Thioredoxins; D014362:Trypsinogen; D001120:Arginine; D004765:Enteropeptidase; D008239:Lysine