Purification of c7 type cytochromes from
Geobactar sulfurreducens
Argonne National LaboratoriesBioscience Division
Summer 2012
Tom Hajek
Overview
What is Geobactar sulfurreducens? What processes do we use to get the desired
protein? Why are we so interested in this particular
protein? Results Long Term Goals
Our FriendGeobactar sulfurreducens
Found in soil and sediments.
“Eats” U and other heavy metals.
Currently being used in bio-remediation of ground water.
Produces MANY multi-heme proteins (cytochromes).
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• Periplasmic cytochrome A (PpcA)
• The most abundant cytochrome in the periplasm of G. sulfurreducens
• Involved in iron (III) reduction and can reduce other metals, e.g. U(VI), Tc(VII)
Cytochrome c7, PpcA
Londer et al., BBA, 1554 (2002), 202-211
What are Cytochromes?
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Cytochromes c: cytochromes with covalently bound heme(s)
Electron transfer proteins that carry heme as a prosthetic group Involved in photosynthesis & aerobic/anaerobic respiration Function is related to the valence change of heme iron, Fe2+ Fe3+
First we must clone....
The gene that codes for PpcA (c7) is cloned into E. coli and selected for by plating with antibiotics
Only the cells that are resistant and contain our gene of interest can grow.
The lac-operon with c7 gene(region of interest)
lac promoter OmpA leader c7 gene (mature)
Y. Londer et al 2002
XbaI HindIII
Role of IPTGRole of IPTG RNA polymerase is
“stuck” on promoter while the repressor is bound to operator.of the lac operon.
IPTG acts as an analogous substrate of allolactase and transcription can begin.
Expression of PpcA in Periplasmic Space
Isolation of Periplasmic Fraction Centrifugation Centrifugation
15min at 4000 rpm 15min at 4000 rpm @ 4@ 4°°C.C.
Re-suspend pellet in Re-suspend pellet in TES buffer (100mM TES buffer (100mM Tris-HCL, pH 7.5, Tris-HCL, pH 7.5, 0.5mM EDTA, 20% 0.5mM EDTA, 20% sucrose)sucrose)
Add protease Add protease inhibitor.inhibitor.
Add lysozyme and Add lysozyme and incubate @ 4incubate @ 4°°C.C.
Cold HOH shock.Cold HOH shock.Centrifuge 15min at Centrifuge 15min at
20K rpm @ 420K rpm @ 4°°C.C.Supernatant = Supernatant =
Periplasmic fraction Periplasmic fraction (this has PpcA in it)(this has PpcA in it)
Cation exchange column
Ionoshpere resin
Protein Purification
CysHis
Typical cytochrome c7
Heme cofactor is covalently attached to the polypeptide through thioether bonds
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10 20 30 C7-1 A D D . I V L K A K N G D V K F P H K A H Q K A V P D C K K C H E . K G P G K I C7-2 A D T . M T F T A K N G N V T F D H K K H Q T I V P D C A V C H G . K T P G K I C7-3 I D K . I T Y P T R I G A V V F P H K K H Q D A L G E C R G C H E . K G P G R I C7-4 A D . V I L F P S K N G A V T F T H K R H S E F V R E C R S C H E . K T P G K I C7-5 H D K V V V L E A K N G N V T F D H K K H A G V K G E C K A C H E T E A G G K I 40 50 60 70 C7-1 E G F G K E M A H G K G C K G C H E E M K K G P T K C G E C H K K - - - - PpcA C7-2 E G F G K E M A H G K S C K G C H E E M K K G P T K C G E C H K K - - - - PpcB C7-3 D G F D K V M A H G K G C K G C H E E M K I G P V R C G D C H K G G S T H PpcC C7-4 R N F G K D Y A H . K T C K G C H E V R G A G P T K C K L C H T G - - - - PpcE C7-5 A G M G K D W A H . K T C T G C H K E M G K G P T K C G E C H K K - - - - PpcD
Aligned sequences of the homologs illustrating the different distribution of charged residues
(acidic residues Asp and Glu shown in red and basic residues Lys and Arg shown in blue).
Insertions and deletions of residues also result in different arrangements in space of the side
chains causing variation in surface electrostatic potential.
PpcA c7-1 amino acid sequence
K = Lysine E = Glutamic Acid
CxxCH at residues 27-31, 51-55, 65-69
E39C, K70C, K29C
71 amino acids. Contains 3 heme
groups. “Pocket” in protein
may accept a photosensitizer.
Cysteine(Cys)
K = Lysine E = Glutamic Acid
18
Heme I
Heme III
Heme IV
K70
K29
E39
Cysteine Mutation sites in PpcA studied (E39C, K29C, K70C)
K = Lysine E = Glutamic Acid
Resultsλ 408 210
No. A1 A2 A1/A221 0.763 1.504 0.507522 1.135 1.857 0.611223 1.625 2.154 0.754324 1.918 2.266 0.846625 1.947 2.308 0.843926 1.715 2.24 0.765427 1.436 2.169 0.661928 1.315 2.167 0.60729 1.328 2.223 0.5971
E39Cλ 408 210
No. A1 A2 A1/A29 0.144 0.404 0.356
10 0.189 0.452 0.417611 0.225 0.474 0.474812 0.26 0.522 0.497913 0.254 0.509 0.499414 0.218 0.484 0.450115 0.175 0.448 0.3917
K29Cλ 408 210
No. A1 A2 A1/A255 0.375 0.795 0.471756 0.593 0.926 0.640557 0.578 0.903 0.640358 0.362 0.709 0.510759 0.188 0.56 0.3363
K70C
Heme:Peptide; 1:1Determined by mass
spectroscopy.A = ε c l
ε = molar extinction coefficientc = concentrationl = path length of cuvette
λ=408nm=heme absorbanceλ=210nm= peptide absorbance
Peak shifts are indicative of heme reduction.
Addition of reducing agent to samples
α
αβ
β
γ
γ
Long-term goalLong-term goal
Combine Chemical and Biological Electron Transfer Function in Metalloprotein Hybrid.
Replace Chemical Solar Catalysts with Bio-hybrid Solar Catalyst.
Create an unlimited supply of clean fuel with minimal input of mechanical energy.
Challenge: Combine Chemical and Biological Electron Transfer Function in Metalloprotein Hybrid
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hv
Catalyst
Photo-sensitizerLinker:
Photosen
sitizer
Catalyst
2H+ H2
2e-
Chemical Solar Catalyst Bio-hybrid Solar Catalyst
hv
hv
e-
fastfast
Problem: Efficiency limited by inability to “re-charge” PS
Solution: Use multi-heme protein frameworks as source for multiple e-
Artero et. al 2008 Ang. Chem. Int. Ed. 47: 564
X
PI: D. Tiede (CSE-ANL)