RESUMO
The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K+ (and Rb+). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter/genética , Hirudinas/genética , Hirudinas/metabolismo , Potássio/metabolismo , RNA Antissenso/análise , Trombina/análise , Animais , Antivirais/análise , Sistema Livre de Células/metabolismo , Fluorometria/métodos , Hirudinas/farmacologia , Luciferases/análise , Luciferases/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA Viral/análise , Coelhos , Rubídio/metabolismo , Trombina/antagonistas & inibidoresRESUMO
The effect of monovalent cation concentrations on the translation was examined in the rabbit reticulocyte cell-free system. The translation of standard reporter gene luciferase was studied using different concentrations of LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and (CH(3))(4)NCl and the acetates of Na(+), K(+), and NH4(+). Only the salts of K(+), Rb(+), and NH4(+) and to some minor extent of Cs(+) significantly supported translation. Optimum concentrations were dependent on the cation used. Optimum concentrations ranged between 40 mM (NH(4)Ac), 80 mM (KCl, NH(4)Cl), and 100 mM (RbCl, KAc). The maximum efficiency of translation depends on the ionic radius of the cation used. KCl and RbCl were superior to all other salts tested in stimulating in vitro translation. The results were confirmed, using a second reporter system, M-hirudin. Here, however, broad optima were observed with RbCl being slightly superior to KCl in supporting translation.
Assuntos
Cátions Monovalentes/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Reticulócitos/metabolismo , Animais , Cátions Monovalentes/química , Sistema Livre de Células , Genes Reporter , Hirudinas/genética , Técnicas In Vitro , Luciferases/genética , CoelhosRESUMO
A simple, highly sensitive, and rapid assay for high-throughput screening of penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through cleavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as various penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate was used for rapid assay of amidase activity in nondenaturing polyacrylamide gels.
Assuntos
Corantes Fluorescentes/metabolismo , Penicilina Amidase/metabolismo , Cumarínicos/síntese química , Cumarínicos/química , Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/genética , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Indicadores e Reagentes/síntese química , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Penicilina Amidase/genética , Penicilina Amidase/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Fatores de TempoRESUMO
A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard thrombin inhibition assays. Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude. This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here. Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin. The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII). The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis. Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner. Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically. Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity. These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity. Beyond that, active hirudin can be displayed on the surface of M13 phage. As a conclusion, applied molecular evolution, i.e. the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.