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The Global ETD Search service is a free service for researchers
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Showing 21 to 22 of 22 (0.021 seconds)Spelling suggestions: "subject:"histag"" "subject:"his.tag""
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Purification of His-tagged Proteins Using WorkBeads 40 TREN as a Pre-Treatment Step Prior Loading Sample onto IMAC Resins with the Purpose to Enhance PerformanceThorsén, Jenny January 2021 (has links)
This work is the result of evaluating a novel strategy for the purification of recombinant His-tagged proteins. Proteins purified in this study were the E. coli translational proteins IF-3, RF-1, and RFF. The study aimed to analyse the potential of using Bio-Works WorkBeads™40 TREN, a multimodal anion ion exchange chromatography resin, as a pretreatment step upstream an immobilized metal ion chromatography (IMAC) resin to enhance performance efficiency of His-tagged protein purification. The method demonstrated here shows potential for anyone seeking to increase the purity of His-tagged protein purification or to introduce an effective purification procedure by replacing a polishing step downstream IMAC with WorkBeads 40 TREN upstream IMAC. The latter contributing to guard the IMAC column from heavy bioburden. This study showed that running WorkBeads 40 TREN prior IMAC captures impurities and removes 97-98 % more dsDNA compared to direct IMAC. WorkBeads 40 TREN is therefore highly advantageous to include early in a purification process to remove protein binding DNA fragments. Moreover, WorkBeads 40 TREN increases purity in the final product by capturing more host cell proteins than when running direct IMAC. This concept is general and WorkBeads 40 TREN could be used upstream a variety of resins such as Protein A and RPC.
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Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code ExpansionOdoi, Keturah 2012 May 1900 (has links)
Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.
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