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Cloning, Sequencing and expression in Escherichia coli of the Rubredoxin gene from Clostridium pasteurianum Mathieu, I., Meyer, J., and Moulis, J. (1992) J. Biochem. 285, (255-262) Background: Structure • • • • Non-heme proteins Composed of 45 to 54 amino acid residues Majority occur in anaerobic bacterium Molecular weight ranging from 5000 and 6000 Daltons Background: Structure Ribbon structure of Rubredoxin from Clostridium pasteurianum showing iron (orange core), and four Cystiene residues. Background: Function •Presumed to serve as electron carriers •Electron-transfer chain in which they participate has only been identified in P. oleovorans Purpose Why study Rubredoxin: • • ETC is important to cellular function Structure is known, but not function Goals: • • Develop a method for over-expression of Rubredoxin Use resultant protein to study role in ETC Cloning Step 1 • • • Derived probes (p1) and (p2) for Rub Digested Cpa genome with RE Southern blotted using p1 and p2 Cloning Step 2 • Determined that Rub DNA appears at 3.9 kb by using gel electrophoresis Cloning Step 3 • • Digested Cpa with RE to isolate Rub sequence Sequence inserted into HindIII-BamHI pUC18 Cloning Step 4 • pUC18 transformed into E.coli DH5alpha cells • Plated on amp plates • • Retested colonies by SB to ensure Rub gene transformed Rub gene was in fact transformed; One clone produced pCPRD1 Sequencing • • • • • • • pCPRD1 sequenced BgLII-SspI no remarkable features ORF1: compared to known reductases ORF2: gene product has no function ORF3: compared to Cpa reductases ORF4: Rubredoxin gene Specific site of Rub gene found Sequenced fragment taken from Cpa Over-Expression • • • Plasmid pCPRD 1 was moved to JM109 E.coli cells Added IPTG to increase expression Did not work Over-Expression • • • Made a second clone, pCPRD2, using specific sites identified on pCPRD1 Plasmid pCPRD2 was moved to JM109 E.coli cells Added IPTG • • Used UV spectroscopy to identify time at which IPTG was most effective: • After 1hr detectable expression • After 4hr leveled off • Stable for at least 24 hrs At optimum time, proteins were harvested Discussion • • • • • Determined that Rubredoxin is generated in one piece Rub function in Cpa still unknown Found no direction connection between ORF1/ORF3 and Rub Even though E.coli does not contain naturally occurring Rub, it is efficient in expressing foreign proteins that have elaborate iron-sulfur clusters Method using Cpa in E.coli and IPTG produces substantially more protein than Cpa Discussion • Amino acid sequence of cloned Rub gene compared to known sequences of Rub and found to have conserved residues. Discussion • Compared UV spec of Rub protein (naturally occurring) to Rub protein (in E.coli) and found them to be the same. Questions? What is ETC?