| Abstract [eng] |
Overexpressed recombinant proteins required for structural genomics are often insoluble. Conventional approaches to obtain soluble, correctly folded protein include low-temperature expression, promoters with different strengths, fusion of the protein with a more soluble partner, coexpression of folding catalyst and chaperones. These approaches do not always work, because they fail to modify the intrinsic folding stability and solubility of the protein. Even when a fraction of the protein is obtained in a soluble form during initial expression or refolding experiments, the protein may still aggregate irreversibly during subsequent workup. In vitro evolution is powerful approach to obtain more soluble protein variants. After protein evolution in vitro more soluble proteins variants from library of mutants can be detected by fusion reporter tags. There is a vast diversity of protein structures and their properties, therefore it is very important to choose for such experiments reporter with good characteristic for genetic screen. Parallel use of few or more alternative reporters in protein evolution experiments may enhance possibility to select more soluble protein variants. In this study was created selection system of more soluble proteins, in which five DNA methyltransferases from different classes were used as fusion reporters. Solubility and activity of methyltransferases was assayed and then appropriate genes of five methyltransferases were fused via flexible linker with gene of insoluble test protein. Expression level, solubility and activity of fused proteins were tested. Practically all fused proteins were insoluble, but they fully methylated plasmid DNA. Assumption was made, that because of high specific activity of methyltransferases minor fraction of soluble fused proteins fully methylate plasmid DNA, that’s why it is essential decrease this activity. For the further experiments was selected one fused protein, which after induction was highly expressed and had least activity of fused methyltransferase. Mutagenesis directed at methyltransferese gene was performed. Two mutants from mutants library with significantly reduced specific activity had been selected. Genes of such mutants were fused via flexible linker with the test insoluble protein. Fused chimeric proteins were insoluble and only slightly methylated DNA. System created during postgraduate studies can be used for selection of more soluble test protein variants. |
