Aptamerek - az antitestek lehetséges alternatívái

Az anitesteken alapuló eljárások számos különböző szerkezeté élelmiszerszennyező szelektív kimutatását teszik lehetővé. Az ellenanyagok szélesköré alkalmazásának alternatíváját jelenthetik az ún. aptamerek, amelyek jól kötődhetnek számos különféle molekulához. A SELEX eljárás elméleti lehetőséget teremt nagy számú aptamer gyors és költséghatékony elåállítására. Az aptamer-célmolekula kölcsönhatás számos analitikai kimutatási módszer alapjául szolgálhat. Mindezek alapján jogosan feltételezhetå, hogy a napjainkban rutinszeréen alkalmazott antitesteken alapuló kimutatási módszerek mellet, illetve helyett a közeli jövåben megjelenhetnek az aptamer alapú élelmiszervizsgálati módszerek is. Methods based on antibodies provide the possibility for the selective identification of many food containants. The so-called aptamers well combined to different molecules could be the alternative. The SELEX procedure provides a theoretical possibility for a rush and cost- efficient production of many aptamers. The interaction between aptamer and molecule can be the basic for many analytical identification methods. In consideration of all that statuments it can be assumed that beside or instead of identification routine methods on the basic of antibodies also food investigation methods on the basic of aptamers will be avaible in the next future

A set of ligation-independent in vitro translation vectors for eukaryotic protein production

<p>Abstract</p> <p>Background</p> <p>The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags.</p> <p>Results</p> <p>We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein.</p> <p>Conclusion</p> <p>Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.</p

Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM

A set of ligation-independent in vitro translation vectors for eukaryotic protein production-3

Oradiography (A). Coomassie Blue staining shows equal loading of MBP (C). MPK6 was either produced in or translated by bilayer method, and 100 ng of purified kinase was used in kinase reactions.<p><b>Copyright information:</b></p><p>Taken from "A set of ligation-independent in vitro translation vectors for eukaryotic protein production"</p><p>http://www.biomedcentral.com/1472-6750/8/32</p><p>BMC Biotechnology 2008;8():32-32.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2311287.</p><p></p

A set of ligation-independent in vitro translation vectors for eukaryotic protein production-1

R (M), wheat germ extract control (C), 1 μl out of 50 μl in vitro translation reaction mixture with pEU3-NII-HLIC (H) or pEU3-NII-GLIC (G) vector. . Bilayer translation reactions with pEU-E01 backbone vector constructs: Molecular weight marker (M), wheat germ extract (C), 5 μl out of 225 μl in vitro translation reaction mixture with pEU-E01-HLIC (H) or pEU-E01-GLIC (G) vector. Proteins present in the translation mixtures were separated on 12% SDS-PAGE gel and detected by Coomassie Blue staining. Vector encoded-kinases are indicated by asterisks.<p><b>Copyright information:</b></p><p>Taken from "A set of ligation-independent in vitro translation vectors for eukaryotic protein production"</p><p>http://www.biomedcentral.com/1472-6750/8/32</p><p>BMC Biotechnology 2008;8():32-32.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2311287.</p><p></p

A set of ligation-independent in vitro translation vectors for eukaryotic protein production-2

Upled kinase (B), MPK6 cleaved from the beads (T), MPK6 cleaved following elution (E). . Purification and cleavage of HisMPK6: Molecular weight marker (M), MagneHis particle coupled kinase (B), MPK6 cleaved from the beads (T), MPK6 cleaved following elution (E). Proteins were translated by CEFC and bilayer method, respectively. A quarter of the total amount of purified protein was separated on 12% SDS-PAGE gel and detected by Coomassie Blue staining.<p><b>Copyright information:</b></p><p>Taken from "A set of ligation-independent in vitro translation vectors for eukaryotic protein production"</p><p>http://www.biomedcentral.com/1472-6750/8/32</p><p>BMC Biotechnology 2008;8():32-32.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2311287.</p><p></p

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence. 2006 The Authors