In this enzyme reaction system, poly (A) was used as a template and oligo (dT) as a primer in addition, DIG-labeled dUTP and biotin-labeled dUTP were incorporated into the complementary DNA (cDNA) during extension, thereby fixing the cDNA onto a streptavidin-coated plate, allowing the use of anti-DIG antibodies to detect reverse transcriptase activity.įigure 1: Diagram of the method used to measure reverse transcriptase activity. Reverse transcriptase activity measurement After synthesis, the integrase was solubilized through the addition of NaCl. After the synthesis, we performed affinity purification using the GST tag that was removed by the PreScission Protease to obtain a purified p51:p66 reverse transcriptase protein complex and integrase (figure 1). One of the layers had a liquid mixture of mRNA, creatine kinase, and wheat germ extract WEPRO7240G the other layer had translation buffer SUB-AMIX SGC. The reverse transcriptase was coexpressed through the p51 and p66 plasmids. We synthesized mRNA from the plasmid DNA template. For tag removal, PreScission Protease recognition sequence was added to p51 and a TEV recognition sequence was added to integrase. We added a GST tag to the N-terminus of the p51 and integrase to allow for affinity purification after synthesis. The p51 and p66 proteins were created to form the reverse transcriptase complex. We created a plasmid DNA template by cloning the reverse transcriptase gene from HIV-1 NL43-strain as well as the integrase gene from HIV-1 HXB2-strain into a pEU expression vector optimized for our wheat cell-free expression system. We were able to synthesize the reverse transcriptase and HIV-1 integrase proteins in vitro. To find a solution for this problem, we evaluated the possibility to synthesize active proteins by using a wheat germ cell-free protein expression system.
However, in some cases, extraction of active proteins using the conventional Escherichia coli or insect cell systems can be difficult because of insolubility. Molecular targets, such as enzymes encoded by viruses, are widely known and are often used for targeted therapy. In order to develop new drugs against viral infections, it is important to understand the mechanisms of viral growth and virulence and to clarify the underlying pathogenesis. Synthesis and activity measurement of viral enzyme proteins Intramolecular interaction analysis Service Intramolecular interaction analysis Service.
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