Purification of three mutant

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PURIFICATION OF THREE MUTANT
CYTOCHROME C7-TYPE PROTEINS FROM
GEOBACTER SULFURREDUCENS.
ABSTRACT: Three mutant forms of periplasmic cytochrome A (PpcA) from Ge-
obacter sulfurreducens were successfully isolated and purified using cation ex-
change chromatography. Spectrophotometer analysis of the samples show evi-
dence that the mature triheme protein was present in the first peak fractions. Mu-
tant versions contain cysteine substitutions at different points of the amino acid se-
quence of PpcA. It is believed that the sulfhydryl group on the cysteine side-chain
could possibly accept a synthetic molecule that would be able to utilize the elec-
tron transfer pathway in the PpcA macromolecule. The mutants that were suc-
cessfully supplied to the collaborators at the Chemistry Division during the course
of the summer 2012 SULI project are named E39C, K29C and K70C.
I. Introduction
PpcA is a triheme protein that is found in the periplasm of Geobacter sulfurreducens. It
consists of 71 amino acids and has three heme prosthetic groups which are covalently bound to
the polypeptide chain via two cysteine thioether bonds. [1]. Mutational studies require the gene
that codes for PpcA to be heterologously overexpressed in Escherichia coli which is the most
common expression host [2]. Although E. coli does code for its own c-type cytochromes, it is
not as efficient at post-translational heme attachment as G. sulfurreducens, therefore total yields
of PpcA that are overexpressed in E. coli are relatively low [2].
II. Materials and Methods.
A. Culture growth/expression. A 50 mL flask containing 2xYT growth medium, am-
picillin (100 μg/mL) and chloramphenicol (34 μg/mL) was inoculated with a single colony of E.
coli that had already transformed with the gene that codes for PpcA. The 50 mL flask was incu-
bated overnight in a shaker at 30⁰C (250 rpm). Four 1L flasks containing 2xYT growth medium
were inoculated with 10 mL of the culture that had been grown overnight, and then incubated at
30⁰C at 250 rpm for 8-10 hours. IPTG was added to each flask to a final concentration of 100
μM and shaking was reduced to 200 rpm and culture was allowed to grow overnight.

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B. Isolation of periplasmic fraction. Cells were harvested by centrifugation with SLC-
4000 rotor for 15 min at 4000 rpm, 4⁰C. Supernatant was removed and pellet was gently re-
suspended with a rubber policeman in 40 mL TES buffer (100 mM Tris-HCl, pH 8.0, 0.5 mM
EDTA, 20% sucrose). Protease inhibitor tablet was added to the suspension. Lysozyme (0.5
mg/mL) was then added to the suspension and incubated at 4⁰C for about one hour. An equal
amount of 4⁰C milli-Q water was added and suspension was left to incubate with gentle shaking
for 15 additional minutes. Suspension was then centrifuged with SS-34 rotor at 20K rpm for 15
min, 4⁰C. The supernatant which is the periplasmic fraction and containing the protein was col-
lected and pellet was discarded.
C. Ion exchange. 1
Standard protocol. Periplasmic fraction was concentrated by loading
onto a cation exchange column containing Ionosphere resin and equilibrated with 10mM Tris-
HCl pH 7.5. and then re-eluted with 10mM Tris-HCl pH 7.5 + 1 M NaCl. We then dialyzed the
prep against 100 fold excess of 10 mM Tris-HCl, pH 7.5. Sample was then centrifuged (30 min,
20K rpm, 4⁰C) and any precipitate was removed. Suspension was reloaded onto the same cation
exchange column and equilibrated with 10 mM Tris-HCl, pH 7.5 then eluted with a 150 mL gra-
dient of 0-300 mM NaCl in 10mM Tris-HCl, pH 7.5 and collected in 3 mL fractions using
FRAC-100 collector. All fractions that had an OD reading ratio A408/A210 closest to 0.8 were
considered electrophoretically pure.
1. K70C was first mixed with a 1:2 ratio with 10mM Tris-HCl, pH 7.0 buffer and centri-
fuged at 20K rpm for 15 min, 4⁰C. Periplasm was loaded onto cation exchange column packed
with Ionosphere resin at 1.2 mL/min. Protein did not bind to column and flowthrough (named
K70C.2) retained a pink color and was saved for further purification steps. Column was washed
with 10mM Tris-HCl, pH 7.0. Flowthrough was diluted further to 3:1 with 10mM Tris-HCl, pH
7.0 buffer (200 mL K70C periplasm + 400 mL Tris-HCl) and loaded onto an exchange column
containing Sp sepharose matrix. Periplasmic fraction was re-eluted using 10mM Tris-HCl, pH
7.5 + 1M NaCl. Flowthrough was then dialyzed in ~100 fold excess 10mM Tris-HCl pH 7.5
overnight. Prep was collected the following morning and centrifuged at 20K rpm for 30 min,
4⁰C. Supernatant was placed into a graduated cylinder and loaded onto a cation exchange col-
umn after diluting 2x with 10mM Tris-HCl, pH 7.5. Both K70C and K70C.2 were ultimately
loaded onto the same cation exchange column containing ~31.4 cm3
ionosphere matrix and
equilibrated with 20mM Tris-HCl, pH 8.0 + 1 mM DTT. Column was eluted with a gradient of
0-100% of buffer B using 20mM Tris-HCl, pH 8.0 + 1mM DTT for buffer A, and 20mM Tris-
HCl, pH 8.0 + 0.5M NaCl for buffer B. Fractions were collected using Frac-100 in 3 mL incre-
ments. Absorbance of the first peak was measured using Shimadzu UV160U UV-visible record-
ing spectrophotometer (Fig. 1-2).
2. E39C periplasm was first dialyzed overnight in excess 10mM Tris-HCl, pH 7.5. Cati-
on exchange column containing Ionosphere was equilibrated with 10mM Tris-HCl, pH 7.5 at 1
mL/min. Periplasm was loaded onto column at 2.0 mL/min. Column was then eluted using
1 Deviations from the Standard protocol developed by Y. Londer are explained in detail under the subheadings K70C,
E39C and K29C respectively

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10mM Tris-HCl, pH 7.5. + 0.5M NaCl at 1 mL/min. Flowthrough (~405 mL) was re-loaded on-
to exchange column and a concentration gradient using a Pharmacia P-500 pump (Run Method
#6) using 10mM Tris-HCl, pH 7.5 for buffer A and 0.5M NaCl for buffer B. Buffer B was
switched to 10mM Tris-HCl, pH 7.5. + 1M NaCl after four hours (Pharmacia pump readings:
Time/Vol = 222.8; Flow Rate= 1.0; %B= 37.0) to speed up elution process. Fractions were col-
lected in 3mL increments using Frac-100 fraction collector when Pharmacia P-500 had the fol-
lowing estimated readings: Time/Vol ~250.0mL; Flow Rate= 1.0; %B = 37<%B<45.5. Absorb-
ance of the first peak was measured using Shimadzu UV160U UV-visible recording spectropho-
tometer (Fig. 3-4).
3. K29C periplasm (~590 mL) was first dialyzed in ~100 fold excess 10mM Tris-HCl,
pH 7.5 overnight. Periplasm was collected from dialysis tubes and centrifuged for 30 min at 20K
rpm, 4⁰C. Supernatant (~900 mL) was collected and pellet discarded. Periplasm was then load-
ed onto cation exchange column containing Ionosphere resin at ~2-3 mL/min. After protein was
bound, column was washed with 1200 mL of 10mM Tris-HCl, pH 7.5. Column was then eluted
with 10mM Tris-HCl, pH 7.5 + 0.5M NaCl at 2.0 mL/min. Flowthrough (~140 mL) was dia-
lyzed overnight in ~100 fold excess 10mM Tris-HCl, pH 7.5. Suspension (~220 mL) was col-
lected from dialysis tubes and centrifuged for 30 min at 20K rpm, 4⁰C. Supernatant was loaded
onto a cation exchange column and washed with 10mM Tris-HCl, pH 7.5. An elution gradient
was done using Pharmacia P-500 pump (run method #6) using 10mM Tris-HCl, pH 7.5 for buff-
er A and 10mM Tris-HCl, pH 7.5. + 0.5M NaCl for buffer B. Protein was moving slowly
through column, so buffer B was switched to 10mM Tris-HCl, pH 7.5 + 1M NaCl. Fractions
were collected in 3 mL increments using Frac-100 fraction collector. Absorbance of the first
peak was measured using Shimadzu UV160U UV-visible recording spectrophotometer (Fig. 5-
6).
III. Results and discussion
K70C. The major peak fractions 55-59 (Fig. 1) were collected in 3 mL increments and
the A408/A210 ratio was analyzed using a Shimadzu® UV160U UV-visible recording
spectrophotometer.
Figure 1. K70C absorbtion at 408/210nm. Shows fraction #57 to have the highest heme/peptide
ratio closest to 1.0.
λ 408 210
No. A1 A2 A1/A2
55 0.375 0.795 0.4717
56 0.593 0.926 0.6405
57 0.578 0.903 0.6403
58 0.362 0.709 0.5107
59 0.188 0.56 0.3363

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Figure 2. K70C analysis of fraction #57 in the oxidized and reduced states. The α peak at 552.0
nm (A= 0.1486) and the red shifted γ peak at 418nm (A= 0.7515) are signatures that show our
triheme protein is present when the sample is reduced with sodium dithionite.
Figure 3. . E39C absorbtion at 408/210nm. Shows fraction #24 to have the highest
heme/peptide ratio closest to 1.0.
λ 408 210
No. A1 A2 A1/A2
21 0.763 1.504 0.5075
22 1.135 1.857 0.6112
23 1.625 2.154 0.7543
24 1.918 2.266 0.8466
25 1.947 2.308 0.8439
26 1.715 2.24 0.7654
27 1.436 2.169 0.6619
28 1.315 2.167 0.607
29 1.328 2.223 0.5971
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
250 400 550
Absorbance
λ nm
K70C Fr#57
K70C-Ox
K70C-Red

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Figure 4. E39C analysis of fraction #24 in the oxidized and reduced states. The α peak at 552.0
nm (A= 0.1750) and the red shifted γ peak at 419.5nm (A= 0.9982) are signatures that show our
Figure 5. K29C absorbtion at 408/210nm. Shows fraction #13 to have the highest heme/peptide
ratio closest to 1.0.
λ 408 210
No. A1 A2 A1/A2
9 0.144 0.404 0.356
10 0.189 0.452 0.4176
11 0.225 0.474 0.4748
12 0.26 0.522 0.4979
13 0.254 0.509 0.4994
14 0.218 0.484 0.4501
15 0.175 0.448 0.3917

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Figure 6. K29C analysis of fraction #13 in the oxidized and reduced states. The α peak at 552.0
nm (A= 0.1882) and the red shifted γ peak at 418.0nm (A= 1.1471) are signatures that show our
Calculations:
1. = mg/mL
2.
An estimated amount of 1-2 mg of each mutant (K70C, E39C and K29C) was supplied to
the collaborators in the Chemistry Division after purification. Although a solution to low effi-
ciency of post-translational modifications (the gene cluster ccmABCDEFG in E. coli that is re-
sponsible for heme attachment of its own c-type cytochromes) has been discovered, this yield is
still lower than the wild type PpcA grown under the same general protocol [2]. This could have
to do with the amount of positive charge lost when lysine is substituted with cysteine. This slight
charge difference may make it more difficult for the mutants K70C and K29C to bind to the ion-
osphere matrix in the cation exchange column. As a result, some of the mature proteins contain-
ing all three prosthetic heme units may have been lost during the binding process and may also
have affected the amount of mature protein that was eluted during the concentration gradient.
Although it is not yet clear, this observation could be supported by the results since the mutant
E39C had a negatively charged glutamic acid residue replaced with a neutral cysteine and had
more affinity to the binding matrix and supplied the highest yield of protein with a peptide/heme
ratio closest to 1.0. Future purification of a new strain of K70C at a higher pH level may in-
crease its binding affinity to the column. Also by dialyzing the periplasm before trying to bind it
to the cation exchange column might get rid of some of the impurities in the periplasmic fraction
and aid in better binding of the protein.

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References
[1] P. Raj Pokkuluri, Yuri Y. Londer, Norma E.C. Duke, W. Chris Long, Marianne Schiffer,
Family of Cytochrome c7-Type Proteins from Geobacter sulfurreducens: Structure of One Cyto-
chrome c7 a1 1.45 Ḁ Resolution. Biochemistry (2004) 1-2.
[2] Yuri Y. Londer, P. Raj Pokkuluri, David M. Tiede, Marianne Schiffer, Production and pre-
liminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurre-
ducens in Escherichia coli. BBA (2002) 1-2.

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Acknowledgments:
I would like to thank the Department of Energy, all of the members of the SULI program,
Argonne National Laboratories, and especially Dr. P. Raj Pokkuluri for giving me the opportuni-
ty to learn about protein purification during my summer 2012 appointment. I would also like to
thank my program advisors at Northern Illinois University for providing me with the LOR’s that
are required to be accepted into the program. And finally my loving wife, whose support and
patients has helped to make it possible for me to work in one of the finest laboratories in the
world.

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