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University of Cincinnati U UNIVERSITY OF CINCINNATI Date: August 17, 2009 I, Mohamed M. Marei , hereby submit this original work as part of the requirements for the degree of: Master of Science in Chemistry It is entitled: The use of pLysB19, a new plasmid, for in vitro transcription of milligram quantities of human lysyl tRNA and purification by urea denaturing PAGE Mohamed M. Marei Student Signature: This work and its defense approved by: Committee Chair: Pearl Tsang Albert Bobst Patrick Limbach Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: Pearl Tsang The use of pLysB19, a new plasmid, for in vitro transcription of milligram quantities of human lysyl tRNA and purification by urea denaturing PAGE A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science in the Department of Chemistry of the College of Arts and Sciences August 2009 by Mohamed M. Marei B.S. University of Cincinnati June 2006 Committee Chair: Pearl Tsang, Ph.D. Abstract A method for production of milligrams of hKtRNA, using new template plasmid pLysB19, by in vitro transcription is presented and compared to methods in use elsewhere. The in vitro transcription yield was found to be almost exclusively dependent on Mg2+ and template concentration. Currently, production of milligrams of hKtRNA is possible but only by use of higher template concentrations than reported elsewhere (7μg vs. 1μg per 10μL reaction). The typical yield of hKtRNA is ≥10μg per 10μl of transcription reaction mixture, of which approximately 6.5μg is recovered after gel purification. Up to 75% of the in vitro transcribed hKtRNA can be aminoacylated by human lysyl tRNA synthetase on acidic urea denaturing PAGE. The requirement of higher template concentrations than reported elsewhere to achieve similar yields is probably due to reduced T7RNAP activity; but the exact cause cannot be stated with absolute certainty due to multiple variations between the in vitro transcription reaction described here and reactions previously described elsewhere. However, recent results by others in this group show lower template concentrations used in the system described here can produce higher yields than shown here when different batches of T7RNAP are used. This is very supportive of conclusions here that reduced T7RNAP activity was the basis for the requirement of higher template concentrations than reported elsewhere. Large-scale production of new template plasmid, pLysB19, is achieved by regeneration and re- use of silica membrane and ion exchange columns provided in commercial kits. Design and construction of pLysB19, done elsewhere, is also described. The transcribed hKtRNA is purified to ~95% on denaturing urea PAGE. The cause of the ~5% degradation products is explored through incubations of hKtRNA in high and low magnesium, as well as high and low pH buffers to determine whether these factors contribute to transcript degradation. The transcript degradation product pattern did not appear to be affected at Mg2+ concentrations as high as 207mM, or during incubations in pH 6 gel elution buffer. These results suggest that degradation under the purification conditions (37 C for 12 hours at pH8) are not due to the reported instability of RNA in the presence of Mg2+ at elevated temperature or pH. A method is also adapted for full resolution of the aminoacylated hKtRNA band from non- aminoacylated hKtRNA on longer acidic urea denaturing PAGE than previously described. A method for comparing the activities of hKRS using a 3H lysine charging assay is also presented and preliminary results are shown. In summary, the results and methods described here may serve as a guide for future experiments which require preparation of milligrams of in vitro transcribed hKtRNA, or the resolution of aminoacylated hKtRNA from non-aminoacylated hKtRNA on acidic urea PAGE. ii iii Acknowledgments I would like to thank my advisor, Dr. Pearl Tsang, for the opportunity to continue my studies, her advice, and many contributions to my scientific training. I would also like to thank Dr. Albert Bobst and Dr. Patrick Limbach for serving on my committee and for their advice. I also thank Dr. H. Brian Halsall for professional and personal advice and for always pushing me to achieve excellence throughout my studies at UC. I also thank the rest of the Dr. Tsang group, both past and present members, for their support- It was a pleasure to get to know and work with all of you. A special thanks to Rehana Siddiqui, Christian Robinson, and Jon Weber for experiments in support of this work. A special thanks to Mike Howell (Protein Express, Inc) for making and providing the pLysB19 plasmid and tons of very helpful technical advice. I also thank Dr. Carol Caparelli (UC) for helpful technical advice and assistance during parts of this research. Thank you to the Dr. Carolyn Price group (UC), especially Benjamin Linger, for providing equipment and training necessary for the aminoacylation assays described here. Thank you to the Dr. Musier-Forsyth group (OSU), especially Christopher Jones, for their collaboration and enthusiasm to help. I am also grateful to the Dr .Stuart Le Grice group (NCI) for providing the T7RNAP used for the in vitro transcriptions described here. I also extend special thanks to my Family and Friends for their unwavering support during my studies- You all have my deepest gratitude. iv Table of Contents List of Figures ........................................................................................................... 4 List of Tables ............................................................................................................ 7 List of Charts ............................................................................................................ 8 List of Abbreviations and Terms ............................................................................ 9 1. Background Lys 1.1 Structure and Function of tRNA3 .............................................................................................. 11 Lys 1.2 Function of tRNA3 Post-transcriptional Modifications ........................................................... 12 2. Preparation of In Vitro Transcription Template 2.1 Introduction 2.1.1 Previously used templates for in vitro transcription of hKtRNA ........................................ 14 2.1.2 Design and construction of a new in vitro transcription template ...................................... 17 2.2 Experimental 2.2.1 Transformation ........................................................................................................................ 20 2.2.2 Quick preps to test for successful transformation ................................................................ 21 2.2.3 Plasmid amplification, isolation, and quantifying yields ...................................................... 21 2.2.4 Regeneration of columns ......................................................................................................... 22 2.2.5 Plasmid Sequencing ................................................................................................................. 22 2.2.6 Restriction Digests ................................................................................................................... 22 2.3 Results & Discussion 2.3.1 Preparation of pLysB19 ..............................................................................................24 2.3.2 Re-use of Plasmid Purification Columns ...................................................................25 2.3.3 Sequencing of pLysB19 ...............................................................................................26 2.3.4 Digesting pLysB19 with BglI ......................................................................................27 2.4 Conclusions .........................................................................................................................28 1 3. Production of hKtRNA by In Vitro Transcription 3.1 Introduction 3.1.1 Transcription Buffer ...................................................................................................29 3.1.2 T7 RNA Polymerase ....................................................................................................32 3.2 Experimental 3.2.1 Determination of optimal Mg2+ concentration ..........................................................34 3.2.2 Determination of optimal Template concentration ..................................................35 3.2.3 Determination of whether Mg2+ replenishment increases transcript yield ............35 3.2.4 Determination of whether BSA or PEG8000 improve transcript yield ..................35 3.2.5 Determination of whether reaction scale affects yield .............................................36 3.2.6 Demonstrative large scale transcriptions ..................................................................36 3.3 Results & Discussion 3.3.1 Effect of Mg2+ Concentration on hKtRNA Yield ......................................................37 3.3.2 Effect of Template Concentration on hKtRNA Yield ..............................................38 3.3.3 Effect of BSA, PEG8000, and Mg2+ Replenishment on hKtRNA Yield .................40 3.3.4 Effect of Reaction Scale on hKtRNA Yield ...............................................................41
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