1. 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)

2. 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

3. Background: Structure Ribbon structure of Rubredoxin from Clostridium pasteurianum showing iron (orange core), and four Cystiene residues.

4. Background: Function Presumed to serve as electron carriers Electron-transfer chain in which they participate has only been identified in P. oleovorans

5. 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

6. Cloning Step 1 Derived probes (p1) and (p2) for Rub Digested Cpa genome with RE Southern blotted using p1 and p2

7. Cloning Step 2 Determined that Rub DNA appears at 3.9 kb by using gel electrophoresis

8. Cloning Step 3 Digested Cpa with RE to isolate Rub sequence Sequence inserted into HindIII-BamHI pUC18

9. 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

10. 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

11. Over-Expression Plasmid pCPRD 1 was moved to JM109 E.coli cells Added IPTG to increase expression Did not work

12. 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

13. 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

14. Discussion Amino acid sequence of cloned Rub gene compared to known sequences of Rub and found to have conserved residues.

15. Discussion Compared UV spec of Rub protein (naturally occurring) to Rub protein (in E.coli) and found them to be the same.

16. Questions?

17. What is ETC?