Transcript Cloning, Sequencing and expression in Escherichia coli of

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
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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:
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ETC is important to
cellular function
Structure is known, but
not function
Goals:
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Develop a method for
over-expression of
Rubredoxin
Use resultant protein
to study role in ETC
Cloning Step 1
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Derived probes (p1) and (p2) for Rub
Digested Cpa genome with RE
Southern blotted using p1 and p2
Cloning Step 2
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Determined that Rub
DNA appears at 3.9
kb by using gel
electrophoresis
Cloning Step 3
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Digested Cpa with
RE to isolate Rub
sequence
Sequence inserted
into HindIII-BamHI
pUC18
Cloning Step 4
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pUC18 transformed into E.coli DH5alpha cells
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Plated on amp plates
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Retested colonies by SB to ensure Rub gene
transformed
Rub gene was in fact transformed; One clone
produced pCPRD1
Sequencing
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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
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Plasmid pCPRD 1
was moved to JM109
E.coli cells
Added IPTG to
increase expression
Did not work
Over-Expression
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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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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
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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
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Amino acid sequence of cloned Rub gene compared to known
sequences of Rub and found to have conserved residues.
Discussion
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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?