Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia; Friedrich-Alexander-Universität Erlangen-Nürnberg, Egerlandstraße 1, Erlangen, Germany.
J Biotechnol. 2013 Dec;168(4):506-10. doi: 10.1016/j.jbiotec.2013.09.019. Epub 2013 Oct 4.
High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.
高水平的重组蛋白表达可能导致不溶性包涵体的形成。这些复杂的聚集体通常在强变性剂中溶解，如 6-8M 尿素，但如果可能的话，在温和条件下溶解可以促进生物活性蛋白的后续复性和纯化。市售的 GST 标签测定法是专为在天然条件下定量测定 GST 活性而设计的。包涵体中积累的 GST 融合蛋白被认为不能通过这种测定法检测到。在这项工作中，使用 4M 尿素进行重组产生的蛋白质的溶解。在 2M 尿素中测定 rGST 的活性，结果表明 rGST 在这种变性条件下保留了 85%的活性。用 1-氯-2,4-二硝基苯（CDNB）进行比色 GST 活性测定，用于快速检测靶向包涵体的表达，并鉴定可以在低浓度变性剂中溶解的包涵体蛋白。通过跟踪两种具有生物制药价值的 GST 融合过敏原（GST-Der p 2 和 GST-Mus a 5）在大肠杆菌中的蛋白表达来评估该测定法的适用性，这两种过敏原都靶向包涵体。
