A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described. E. coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot. Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability. Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E. coli RNase HI. Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available. Study holds ProTherm entries: 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 13382, 13383, 13384, 13385, 13386, 13387, 13388, 13389, 13390, 13391, 13392, 13393, 13394, 13395, 13396, 13397, 13398, 13399, 13400, 13401, 13402, 13403, 13404, 13405, 13406, 13407, 13408, 13409, 13410, 13411 Extra Details: Activity determined at 30 degree C Escherichia coli ribonuclease HI; thermostability ;,mutagenesis; protein stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
|Number of data points||204|
|Proteins||Ribonuclease HI ; Ribonuclease pancreatic ; Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: activity pH:3.0, temp:50.1 C, buffers:glycine-HCl: 10 mM ; Experimental Assay: ddG pH:3.0, temp:50.1 C, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity temp:52.7 C, buffers:Sodium acetate: 20 mM, pH:5.5 ; Experimental Assay: ddG temp:52.7 C, buffers:Sodium acetate: 20 mM, pH:5.5 ; Experimental Assay: activity pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: Tm pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHvH pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: Tm pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: dHvH pH:5.5, buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm pH:3.0, buffers:glycine-HCl: 10 mM ; Derived Quantity: dTm pH:5.5, buffers:Sodium acetate: 20 mM|
|Libraries||Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|