Cloning, Sequencing and expression in Escherichia coli of the Rubredoxin gene from Clostridium pasteurianum

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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)

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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

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

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

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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

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Cloning Step 1

• Derived probes (p1) and (p2) for Rub
• Digested Cpa genome with RE
• Southern blotted using p1 and p2

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Cloning Step 2

• Determined that Rub DNA appears at 3.9 kb by
  using gel electrophoresis

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Cloning Step 3

• Digested Cpa with RE to isolate Rub sequence
• Sequence inserted into HindIII-BamHI pUC18

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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

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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
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Over-Expression

• Plasmid pCPRD 1 was moved to JM109 E.coli cells
• Added IPTG to increase expression
• Did not work

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Over-Expression

• 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

• Made a second clone, pCPRD2, using specific sites
  identified on pCPRD1
• Plasmid pCPRD2 was moved to JM109 E.coli cells
• Added IPTG

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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

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Discussion

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

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Discussion

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

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Questions?
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What is ETC?