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• W2112748367 abstract "The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIapro proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the ω subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the λcI repressor, containing an NIapro cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIapro proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds. The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIapro proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the ω subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the λcI repressor, containing an NIapro cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIapro proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds. Selective inhibition of protein activity inside the cell is of fundamental importance for the investigation of biological processes as well as for the drug discovery process. Experimental approaches to achieve this goal have become increasingly available through the use of genetic knockouts and small interfering RNA-mediated knockdown of target proteins (1Dove A. Nat. Biotechnol. 2002; 20: 121-124Crossref PubMed Scopus (68) Google Scholar). However, these techniques knock out the expression of the entire gene of interest and are thus not able to discriminate, e.g. between the different functions of protein variants originating from the same gene (2Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1775) Google Scholar). Moreover, the effect mediated by RNA interference is often only weak, especially if the cellular stability of the protein of interest is high, as only the de novo synthesis is (partially) inhibited. Recent studies have also demonstrated that RNA interference effects are not always specific for the targeted gene (3Sledz C.A. Holko M. de Veer M.J. Silverman R.H. Williams B.R. Nat. Cell Biol. 2003; 5: 834-839Crossref PubMed Scopus (1225) Google Scholar, 4Jackson A.L. Bartz S.R. Schelter J. Kobayashi S.V. Burchard J. Mao M. Li B. Cavet G. Linsley P.S. Nat. Biotechnol. 2003; 21: 635-637Crossref PubMed Scopus (1927) Google Scholar, 5Bridge A.J. Pebernard S. Ducraux A. Nicoulaz A.L. Iggo R. Nat. Genet. 2003; 34: 263-264Crossref PubMed Scopus (856) Google Scholar). The use of inhibitory molecules directly acting at the protein level is thus a complementary approach. This strategy allows the targeting of single functions residing in different domains of multidomain protein complexes or those due to post-translational modifications. An important consideration is also that such proteinaceous inhibitors may be used in the subsequent characterization of the corresponding target protein in vitro or may even serve as first leads in the drug discovery process. Intracellular proteinases are important regulators of signal transduction, RNA transcription, cell cycle progression, apoptosis, and development (6Sices H.J. Kristie T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2828-2833Crossref PubMed Scopus (30) Google Scholar, 7Vanaman T.C. Bradshaw R.A. J. Biol. Chem. 1999; 274: 20047Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and many other processes, and they are also used by viruses in the processing of polyprotein precursors (8Dougherty W.G. Semler B.L. Microbiol. Rev. 1993; 57: 781-822Crossref PubMed Google Scholar). To elucidate their function early in the discovery process, specific small molecule inhibitors will usually not be available, and thus a rapid approach to generate specific inhibitors that function inside the cell would be very valuable. One way of approaching this challenge would be to use artificial proteinase inhibitors based on proteins. Although a large number of protein families have been used by nature for this purpose (10Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (468) Google Scholar), the great majority of these inhibitors are secreted proteins and contain disulfide bonds. Thus, they work naturally on secreted proteinases and, consequently, have been re-engineered to target extracellular proteinases (9Skerra A. J. Mol. Recognit. 2000; 13: 167-187Crossref PubMed Scopus (203) Google Scholar). Even though there are also natural intracellular proteinase inhibitors controlling many of the processes mentioned above (10Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (468) Google Scholar), they have not been used as scaffolds for deriving new specificities up to now. Another approach would be to use scaffolds that are not derived from proteinase inhibitors for this purpose. The generation of novel inhibitors is difficult, because polypeptides are first and foremost substrates of proteinases. The challenge is thus to achieve selective binding without cleavage or by maintaining a stable complex between proteinase and inhibitor even after cleavage of the latter. An antibody scFv fragment that works in the reducing intracellular milieu (11Sun J. Pons J. Craik C.S. Biochemistry. 2003; 42: 892-900Crossref PubMed Scopus (38) Google Scholar) has been reported for this purpose. However, because these molecules also rely on disulfide bonds for stability (12Biocca S. Ruberti F. Tafani M. Pierandrei-Amaldi P. Cattaneo A. Bio/Technology. 1995; 13: 1110-1115Crossref PubMed Scopus (152) Google Scholar, 13Ewert S. Honegger A. Plückthun A. Methods (San Diego). 2004; 34: 184-199Crossref PubMed Scopus (182) Google Scholar, 14Jobling S.A. Jarman C. Teh M.M. Holmberg N. Blake C. Verhoeyen M.E. Nat. Biotechnol. 2003; 21: 77-80Crossref PubMed Scopus (156) Google Scholar), they may not provide a general solution for this kind of application. We therefore wished to investigate whether another class of proteins, repeat proteins, can be engineered to act as proteinase inhibitors. We previously reported the generation of designed ankyrin repeat proteins (DARPins) 3The abbreviations used are: DARPin, designed ankyrin repeat protein; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-β-d-thiogalactopyranoside; NIapro, potyvirus nuclear inclusion-a proteinase (25 kDa, not containing the viral genome-linked protein domain VPg); RNAP, RNA polymerase; SPR, surface plasmon resonance; TEV, tobacco etch virus; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. as specific binding molecules, and we also showed that they can be selected for intracellular enzyme inhibition, demonstrated for a bacterial kinase (15Forrer P. Stumpp M.T. Binz H.K. Plückthun A. FEBS Lett. 2003; 539: 2-6Crossref PubMed Scopus (125) Google Scholar, 16Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (535) Google Scholar, 17Amstutz P. Binz H.K. Parizek P. Stumpp M.T. Kohl A. Grütter M.G. Forrer P. Plückthun A. J. Biol. Chem. 2005; 280: 24715-24722Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), but it was unclear whether they would contain the properties required for proteinase inhibition. Repeat proteins constitute the largest group of natural proteins specialized in binding. They can be found across all phyla, in the intra- and extracellular space, mediating a diverse set of biological functions (18Bork P. Proteins. 1993; 17: 363-374Crossref PubMed Scopus (446) Google Scholar, 19Andrade M.A. Perez-Iratxeta C. Ponting C.P. J. Struct. Biol. 2001; 134: 117-131Crossref PubMed Scopus (477) Google Scholar, 20Kobe B. Kajava A.V. Curr. Opin. Struct. Biol. 2001; 11: 725-732Crossref PubMed Scopus (1298) Google Scholar). DARPins feature consecutive homologous structural units (repeats) of 33 amino acids, which stack to build up a single folded polypeptide. The elongated repeat domain can be of variable size, depending on the number of repeats, and it displays a rather rigid target-binding surface that can accommodate many different surface residues adaptable to specifically bind a wide range of targets. By structural and sequence consensus analysis of this modular architecture of natural repeat proteins, we constructed highly diverse combinatorial DARPin libraries (15Forrer P. Stumpp M.T. Binz H.K. Plückthun A. FEBS Lett. 2003; 539: 2-6Crossref PubMed Scopus (125) Google Scholar, 21Forrer P. Binz H.K. Stumpp M.T. Plückthun A. Chembiochem. 2004; 5: 183-189Crossref PubMed Scopus (87) Google Scholar, 22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar). These libraries consist of an N-terminal capping repeat, a defined number (typically 2 or 3) of engineered randomized internal repeats, and a C-terminal capping repeat (denoted an N2C and an N3C library; Fig. 1) in a single protein chain, and the molecules assume the ankyrin fold. The theoretical diversity exceeds 1014 for the N2C library and 1023 for the N3C library (22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar). Unselected members of these libraries show very favorable biophysical properties (16Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (535) Google Scholar, 22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar, 23Kohl A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1700-1705Crossref PubMed Scopus (235) Google Scholar), and selected members interact with their target molecules via their randomized positions in a highly specific manner (16Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (535) Google Scholar, 24Kohl A. Amstutz P. Parizek P. Binz H.K. Briand C. Capitani G. Forrer P. Plückthun A. Grütter M.G. Structure (Lond.). 2005; 13: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). DARPins do not rely on disulfide bonds for their stability, nor do they contain free cysteines. Furthermore, they do not show structural similarities to known naturally occurring proteinaceous inhibitors. Here, we investigated whether DARPins can be selected to inhibit the main proteinase responsible for virus maturation of an agriculturally important plant virus, the NIapro proteinase of tobacco etch virus (TEV). NIapro is the main proteinase of potyvirus; it is responsible for two-thirds of all cleavage reactions occurring during the viral infection cycle, and its functionality is vital for successful virus propagation (25Carrington J.C. Dougherty W.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3391-3395Crossref PubMed Scopus (173) Google Scholar, 26Carrington J.C. Dougherty W.G. J. Virol. 1987; 61: 2540-2548Crossref PubMed Google Scholar, 27Carrington J.C. Dougherty W.G. Virology. 1987; 160: 355-362Crossref PubMed Scopus (54) Google Scholar). Our aim to identify DARPin-based proteinase inhibitors was further encouraged by recent findings that naturally occurring proteinase inhibitors could mediate resistance against potyviruses in transgenic plants (28Gutierrez-Campos R. Torres-Acosta J.A. Saucedo-Arias L.J. Gomez-Lim M.A. Nat. Biotechnol. 1999; 17: 1223-1226Crossref PubMed Scopus (145) Google Scholar). In addition, NIapro is structurally highly homologous to the 3C proteinases of the picornavirus family, which are the major cause of numerous human diseases worldwide (29Wang Q.M. Prog. Drug Res. 2001; (Spec. No.): 229-253PubMed Google Scholar). Furthermore, this proteinase can be expressed in functional form in Escherichia coli without harm to the cell, as its highly specific cleavage reaction does not seem to destroy vital E. coli proteins. To accomplish our task, we applied a two-step approach of in vitro selection for binding, followed by in vivo activity screening. Although many assays exist to study proteinase activity in vitro (30Billich A. Hammerschmid F. Winkler G. Biol. Chem. Hoppe-Seyler. 1990; 371: 265-272Crossref PubMed Scopus (52) Google Scholar, 31Matayoshi E.D. Wang G.T. Krafft G.A. Erickson J. Science. 1990; 247: 954-958Crossref PubMed Scopus (562) Google Scholar, 32Richards A.D. Phylip L.H. Farmerie W.G. Scarborough P.E. Alvarez A. Dunn B.M. Hirel P.H. Konvalinka J. Strop P. Pavlickova L. Kostka V. Kay J. J. Biol. Chem. 1990; 265: 7733-7736Abstract Full Text PDF PubMed Google Scholar) and in vivo (6Sices H.J. Kristie T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2828-2833Crossref PubMed Scopus (30) Google Scholar, 33Dasmahapatra B. DiDomenico B. Dwyer S. Ma J. Sadowski I. Schwartz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4159-4162Crossref PubMed Scopus (21) Google Scholar, 34Smith T.A. Kohorn B.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5159-5162Crossref PubMed Scopus (30) Google Scholar), these assays either need purified protein or they lack ease of handling. Therefore, we adapted a known bacterial two-hybrid system (35Dove S.L. Hochschild A. Genes Dev. 1998; 12: 745-754Crossref PubMed Scopus (124) Google Scholar) to serve as an in vivo proteinase activity screen. We were able to select and characterize in vivo active proteinaceous DARPin NIapro inhibitors. The potential of DARPins as a basis for proteinase inhibition and as a general intracellular target validation tool is discussed. Molecular Biology—Unless stated otherwise, all experiments were performed according to protocols of Sambrook et al. (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Enzymes and buffers were from New England Biolabs (Beverly, MA) or Fermentas (Vilnius, Lithuania). All PCRs were performed using the proofreading PfuTurbo polymerase (Stratagene). Plasmids—Plasmids used in this study are listed in Table 1, and their construction is described in detail in the Supplemental Material. The sequences of all inserts in plasmids that were generated by PCR were confirmed by DNA sequencing.TABLE 1Plasmids The abbreviations used are as follows: ApR, ampicillin-resistant; CmR, chloramphenicol-resistant; KanR, kanamycin-resistant; TcR, tetracycline-resistant; gpD, bacteriophage λ coat protein D.PlasmidRelevant detailsSource/Ref.pBRcI-ωApR, ColE1, encodes λcI-wt(residues 1—236)-2Ala-ω(residues 1—90)35pBRcI-T-ωApR, ColE1, encodes λcI-wt(residues 1—236)-linker(NIapro recognition site plus His6 tag)-ω(residues 1—90)This workpMAKcI-T-ω-pDApR, ColE1, encodes λcI-wt(residues 1—236)-linker(NIapro recognition site plus His6 tag)-ω(residues 1—90) under control of Pbla, gpD under control of PT5/lacThis workpMAKcI-T-ω-DLApR, ColE1, encodes λcI-wt(residues 1—236)-linker(NIapro recognition site plus His6 tag)-ω(residues 1—90) under control of Pbla, DARPin library under control of PT5/lacThis workpQI-pDApR, ColE1, RGS- His6 tag-gpD under control of PT5/lac39pZA55-TEVTcR, p15A, encodes NIapro proteinase catalytic domainaAll experiments were performed with a mutant form (S219V) of the C-terminal proteolytic domain of the full-length NIapro protein of TEV. Thus, the proteinase used here lacks its N-terminal VPg domain and is resistant to autoinactivation by truncation of its C-terminal tail (S219N) under control of PBADThis workpAT223_TEVApR, ColE1, encodes NIapro proteinase catalytic domain (S219N) as C-terminal fusion of gpD under control of PT5/lac, N-terminal Avi tag for biotinylationThis workpAT223ApR, ColE1, encodes gpD under control of PT5/lac, N-terminal Avi tag for biotinylationThis workpRK793ApR, ColE1, encodes NIapro proteinase catalytic domain (S219N)as C-terminal fusion of maltose-binding protein under control of Ptac38pBirAcmCmR, p15A, used for in vivo biotinylation; contains birAAvidity, Denver, COpMAKcI-T-ω-DL tagpMAKcI-T-ω-DL analogue, DARPin library member devoid of its N-terminal RGS-His6 tagThis workpQE60ApR, ColE1, PT5/lac-controlled expression plasmidQiagen, Hilden, GermanypBADGFPAC2ApR, ColE1, encodes GFP under control of PBAD57pZA21KanR, p15A, PLtet-O1-controlled expression plasmid37pTRGTcR, ColE1; “dummy” plasmid to confer tetracycline resistance to cells during in vivo screening experiments (control)45a All experiments were performed with a mutant form (S219V) of the C-terminal proteolytic domain of the full-length NIapro protein of TEV. Thus, the proteinase used here lacks its N-terminal VPg domain and is resistant to autoinactivation by truncation of its C-terminal tail Open table in a new tab The vector for the expression of NIapro proteinase in all in vivo experiments, pZA55-TEV, was constructed by inserting the PCR-amplified araC gene plus the PBAD promoter sequence into pZA21-TEV. In turn, pZA21-TEV was constructed by inserting the PCR-amplified gene of the catalytic domain, NIapro, into pZA21 (37Lutz R. Bujard H. Nucleic Acids Res. 1997; 25: 1203-1210Crossref PubMed Scopus (1238) Google Scholar), thereby replacing its KpnI/BamHI fragment. The gene of the catalytic domain NIapro was amplified from pRK793 (38Kapust R.B. Waugh D.S. Protein Sci. 1999; 8: 1668-1674Crossref PubMed Scopus (765) Google Scholar). pBRcI-T-ω is a derivative of pBRcI-ω (35Dove S.L. Hochschild A. Genes Dev. 1998; 12: 745-754Crossref PubMed Scopus (124) Google Scholar) containing a NIapro cleavage site. pMAKcI-T-ω-pD is a derivative of pQI-pD (39Stumpp M.T. Designing Repeat Proteins: From Leucine-rich Repeat Proteins to Ankyrin Repeat Proteins. University of Zürich, Zürich, Switzerland2004Google Scholar) and constitutively expresses the λcI-T-ω fusion protein under control of the β-lactamase promoter Pbla. pMAKcI-T-ω-DL is a derivative of pMAKcI-T-ω-pD in which the pool of DARPins enriched by ribosome display, binding NIapro, replaces phage λ protein D (gpD). Expression of the DARPin pool is under control of the IPTG-inducible promoter PT5/lac. It was used in all in vivo screening experiments. The open reading frames of the ribosome display-selected DARPins were digested with NcoI and HindIII and ligated into pMAKcI-T-ω-pD, yielding the selection plasmid ready for in vivo screening. The pMAKcI-T-ω-DL tag is a derivative of pMAKcI-T-ω-DL and was used for the DARPins 9_1s, 13_1b, 20_2b, and E2_5 in the Western blot experiments. In this vector the DARPin genes lack their N-terminal RGS-His6 tag. pAT223-TEV is a derivative of pAT223 and was used for the expression of His-tagged biotinylated and nonbiotinylated gpD-NIapro fusion protein. Protein Production and Purification—The biotinylated fusion protein pD-NIapro and biotinylated pD alone (plasmids pAT223_TEV and pAT222) were produced by in vivo biotinylation of the N-terminal Avi tag (40Schatz P.J. Bio/Technology. 1993; 11: 1138-1143Crossref PubMed Scopus (511) Google Scholar) by co-expression of BirA from the plasmid pBirAcm in E. coli XL-1 Blue (Stratagene, La Jolla, CA) according to the protocols of Avidity (Denver) and Qiagen (Hilden, Germany). Efficient biotinylation was confirmed by ELISA and Western blotting using a streptavidinalkaline phosphatase conjugate as detection agent (Roche Applied Science) and by mass spectrometry. Nonbiotinylated pD-NIapro for the ELISA analysis was produced in the same way as the DARPin proteins (22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar) using pAT223_TEV in E. coli XL-1 Blue. His tag purification of all proteins was carried out as described (22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar). In Vitro Selection with Ribosome Display—The DARPin library generation has been described (22Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (455) Google Scholar). In this study, an N2C and an N3C DARPin library were used, encoding DARPins consisting of a constant N-terminal capping repeat, two or three internal designed ankyrin repeats, respectively, containing randomized residues, and a constant C-terminal capping repeat as a continuous polypeptide chain. The PCR-amplified libraries in the ribosome display format were transcribed in vitro, and four standard ribosome display selection rounds were carried out as described (16Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (535) Google Scholar, 41Hanes J. Plückthun A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4937-4942Crossref PubMed Scopus (907) Google Scholar). In Vivo Screening—The pools of NIapro binders selected by ribosome display from selection round three and four, starting from the N2C and the N3C library, were combined and ligated into pMAKcI-T-ω-pD, thereby replacing phage λ protein D (gpD) and generating pMAKcI-T-ω-DL, co-introduced with pZA55-TEV into E. coli KS1ΔZ (35Dove S.L. Hochschild A. Genes Dev. 1998; 12: 745-754Crossref PubMed Scopus (124) Google Scholar) and plated on LB agar plates containing 1% glucose, 50 μg/ml ampicillin, 20 μg/ml tetracycline, 0.2% arabinose, 20 μg/ml X-gal, and 20-25 μm IPTG. Cells were grown at 30 °C overnight and checked for blue color development after various times. pMAKcI-T-ω-DL clones were isolated from different blue colonies and re-introduced together with pZA55-TEV into fresh E. coli KS1ΔZ cells, and the screening step was repeated to confirm the phenotype and to eliminate false positives. The DNA of those clones confirmed twice as positive was sequenced using standard DNA sequencing. Size-exclusion Chromatography—Immobilized metal ion affinity chromatography-purified DARPins were analyzed on a Superdex 200 HR gel-filtration column (Amersham Biosciences) at room temperature using a SMART chromatography system (Amersham Biosciences) at a flow rate of 60 μl/min. TBS150 (50 mm Tris-HCl, pH 7.4, 150 mm NaCl) was used as running buffer. ELISA—The biotinylated antigens (pD or pD-NIapro) were immobilized as follows: neutravidin (66 nm, 100 μl/well; Pierce) in TBS150 was immobilized on a Maxisorp plate (Nunc, Roskilde, Denmark) by overnight incubation at 4 °C. The wells were then blocked with 300 μl of 0.5% bovine serum albumin (Fluka, Buchs, Switzerland) in TBS150 for 1 h at room temperature. Biotinylated antigen (100 μl; 1 μm) in TBS150 with 0.5% bovine serum albumin was allowed to bind for 1 h at 4°C. To test whether the binding of the selected DARPins was specific for NIapro, 100 μl of purified DARPins (1 μm) were applied to wells with or without immobilized antigen for 1 h at room temperature. After extensive washing with TBS150, binding was detected with an anti-RGS-His antibody (Qiagen; detects only the N-terminal RGS-His6 tag of the DARPin but not the internal (pD-NIapro) or C-terminal (pD) His6 tag of the antigen), an anti-mouse-IgG-alkaline phosphatase conjugate (Pierce), and p-nitrophenyl phosphate (Fluka). For competition ELISA, the purified DARPins were incubated with 5 μm of free NIapro prior to and during (4 °C, 100 min) the binding reaction. Surface Plasmon Resonance—SPR was measured using a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden). The running buffer was 20 mm HEPES, pH 7.4, 150 mm NaCl, 0.005% Tween 20. A streptavidin SA chip (BIAcore) was used with 2000 response units of biotinylated pD-NIapro immobilized. The interactions were measured at a flow of 50 μl/min with a 5-min buffer flow, a 5-min injection of NIapro binding DARPins in varying concentrations (45 nm to 100 μm), and a dissociation step of 10 min with buffer flow. The signal of an uncoated reference cell was subtracted from the measurements. The equilibrium data of the interaction were evaluated with a global fit using BIAevaluation 3.0 (BIAcore), Scrubber (BioLogic software, Campbell, Australia), and Clamp (42Myszka D.G. Morton T.A. Trends Biochem. Sci. 1998; 23: 149-150Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Western Blot Analysis—Prior to Western blot analysis, antigenic samples were normalized to cell density (A600), and proteins were separated by standard 15% SDS-PAGE. Western blot analysis was done following the protocol of Ref. 43Gallagher S. Winston S.E. Fuller S.A. Hurrell J.G.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York2004: 10.18.11-10.18.24Google Scholar. An Immobilon-P transfer membrane (Millipore, Billerica, MA) was used for sample transfer using semi-dry electroblotting. Sample detection was achieved using an anti-tetra-His antibody (Qiagen) and an anti-mouse-IgG-alkaline phosphatase conjugate (Pierce) or an anti-cI antibody (Invitrogen) and an anti-rabbit-IgG-horseradish peroxidase conjugate (Sigma). Blots were developed using nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate as substrates or chemiluminescent horseradish peroxidase substrate (Millipore). In Vitro Inhibition Study—NIapro activity assays were performed essentially according to published procedures (44Stennicke H.R. Salvesen G.S. Methods (San Diego). 1999; 17: 313-319Crossref PubMed Scopus (160) Google Scholar) with small modifications. Briefly, NIapro (3 μm) and selected DARPins or DARPin E2_5 (300 μm) were preincubated in NIapro reaction buffer (50 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 1 mm dithiothreitol) at room temperature for 5 min prior to starting the measurement. The reaction was started by adding 7.5 μl of 200 μm substrate (Ac-TENLYFQ-amc, where amc is 7-amino-4-methyl-coumarin) to the reaction mixture (Vfinal, 100 μl; cfinal substrate, 15 μm), and initial rates of substrate hydrolysis were immediately recorded by fluorometric measurement of the emission intensity. The assay was carried out at room temperature. The excitation wavelength was 360 nm, and the emission wavelength was 465 nm. Initial velocity data of substrate hydrolysis in the presence of the selected DARPins or E2_5 were normalized to initial velocity data obtained from measurements without DARPins; the value obtained here was arbitrarily set to 100%. All experiments were at least done in triplicate. To obtain NIapro inhibitors from large combinatorial DARPin libraries (15Forrer P. Stumpp M.T. Binz H.K. Plückthun A. FEBS Lett. 2003; 539: 2-6Crossref PubMed Scopus (125) Google Scholar), we chose to follow a two-step procedure consisting of an in vitro selection step to obtain pools of DARPins able to bind NIapro, followed by an in vivo screening step to identify those DARPins which not only bind but also inhibit the proteinase activity. This genetic screen is based on the well characterized transcriptional activation properties of fusion proteins consisting of the ω subunit of E. coli RNA polymerase (RNAP) connected covalently to a DNA-binding protein (bacteriophage λ repressor cI (35Dove S.L. Hochschild A. Genes Dev. 1998; 12: 745-754Crossref PubMed Scopus (124) Google Scholar)). A Genetic Activity Screen for Site-specific Proteolytic Enzymes— The bas" @default.
• W2112748367 created "2016-06-24" @default.
• W2112748367 creator A5025889881 @default.
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• W2112748367 date "2006-12-01" @default.
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• W2112748367 title "Isolation of Intracellular Proteinase Inhibitors Derived from Designed Ankyrin Repeat Proteins by Genetic Screening" @default.
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• W2112748367 doi "https://doi.org/10.1074/jbc.m602506200" @default.
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