Tremblay, Robin Lee (2000) Expression and characterisation of a gene encoding RbpD, an RNA-Binding protein in Anabaena sp. strain PCC 7120. Masters thesis, Memorial University of Newfoundland.
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The RNA-binding protein RbpD, from the cyanobacterium Anabaena sp. Strain PCC 7120 was expressed in Escherichia coli and successfully purified using the IMPACT I system (New England Biolabs). The rbpD gene was cloned into the pCYBl expression vector by using polymerase chain reaction to introduce Ndel and Sapl restriction sites at the 5' end 3' ends of the gene respectively. The 3'-end mutagenesis also changed the stop codon into a cysteine codon. The resulting gene encoded a fusion protein consisting of RbpD, the Saccharomyces cerevisiae VMA intein and a chitin binding domain. Expression of the fusion protein was observed in E. coli strain MCI 061 but Western blot analysis using an intein-directed antibody indicated that significant in vivo intein-directed splicing of the fusion protein occurred. We were unable to eliminate this problem; no fusion protein expression was observed in 8 other E. coli strains tested. Wild-type RbpD was purified following binding of the fusion protein to a chitin column and overnight cleavage in the presence of a reducing agent, dithiothreitol. A number of modifications to the manufacturer's purification protocol were found to be necessary for successful purification. The NaCl concentration in the cleavage and elution buffer was increased from 50 mM to 500 mM to eliminate problems of RbpD solubility. An increase in the dithiothreitol concentration of the cleavage buffer from 30 mM to 50 mM was required for full cleavage. -- A modified form of RbpD containing an hexa-histidine tag in loop 3 of the RNA recognition motif, RbpDl, was also successfully purified. The rbpDl gene, previously constructed by Cynthia Slade, was cloned into the pTRC99A expression vector. An Ncol site was introduced at the 5'-end of the gene using site-directed mutagenesis. This modification also changed the second codon of the gene from serine to alanine. The RbpDl protein was expressed in E. coli strain BL21(DE3)pLysS following induction with IPTG and purified using a nickel-NTA agarose affinity column. The protein was eluted with 100 mM imidazole and appeared to be pure upon analysis using polyacrylamide gel electrophoresis. -- The RNA-binding activity of RbpD and RbpDl were first determined using Sepharose-4B-, polyacrylhydrazido-agarose-, or agarose-bound RNA homopolymers. Both proteins bound strongly to poly(U), less strongly to poly(G), weakly to poly(A), and not at all to poly(C). This pattern is consistent with that observed for other cyanobacterial RNA-binding proteins. There was no apparent difference in the binding affinities of RbpD and RbpDl indicating that the presence of the 6x-histidine tag had no effect. Experiments to detect binding between RbpD and a conserved sequence element in the 5'- untranslated region of rbpD using both electrophoretic mobility shift assays and nitrocellulose filter binding were unsuccessful. Similarly, attempts to detect binding between RbpD and size-fractionated radioactively labelled poly(U) by electrophoretic mobility shift assays were unsuccessful. Two SELEX experiments were also unsuccessful. In both cases, no increase in specific binding over background was detected through four rounds of selection.
|Item Type:||Thesis (Masters)|
|Additional Information:||Bibliography: leaves 158-173.|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Anabaena--Genetics; Cyanobacteria; RNA-protein interactions|
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