Chaperonin Protocols (Methods in Molecular Biology) - Christine Schneider, medicalheaven radiology

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Methods in Molecular Biology
T
TM
VOLUME 140
Chaperonin
Edited by
Christine Schneider
HUMANA PRESS
Methods in Molecular Biology
Chaperonin
Protocols
Protocols
Edited by
Christine Schneider
Purification of Archaeal Chaperonin
1
1
Purification of Archaeal Chaperonin
from Sulfolobus shibatae
Elsie Quaite-Randall and Andrzej Joachimiak
1. Introduction
Sulfolobus shibatae
is a hyperthermophilic archaeon that was first identified
living in acidic geothermal hot springs. This organism grows optimally at pH
3.0–4.0 and 83°C
(1)
, however it grows over the temperature range of 75–
85°C. When
S. shibatae
is subjected to higher temperatures (85–90°C) a heat-
shock response is observed and the major protein induced is a large ring
structure TF55
(2)
, also called archaeosome
(3)
or rosettasome
(4)
, which is
composed of two different subunits
(4)
, designated _ and `
(3
,
4)
. Apart from
the presence of two subunits, another difference between this molecule and the
prokaryotic GroEL is the number of subunits per ring. In
S. shibatae
, nine
subunits form each ring compared with seven in GroEL.
The similarity of this double ring
(2
,
3)
to that of GroEL, together with the
fact that the subunits were 60 kDa, it was a heat-shock protein, and it was
active in protein folding, suggested that it was the archaeal chaperonin
(2)
and
was similar in function to GroEL. However, comparison of the primary struc-
ture showed that both subunits were more closely related to the TCP-1 family
of polypeptides, the eukaryotic cytosolic chaperonin
(2
,
4
,
5)
. In contrast to the
archaeal chaperonin, the eukaryotic cytosolic chaperonin comprises eight
sequence-related polypeptides, which also form the characteristic “double
doughnut.” The archaeal chaperonin therefore gives us a simplified version of
the eukaryotic chaperonin by which it may be possible to determine character-
istics of the eukaryotic chaperonin. Archaeal and mammalian cytoplasmic
chaperonins are often referred to as Type II chaperonins. The chaperonin from
S. shibatae
is a Type II chaperonin
(6)
and is related, by primary sequence, to
From: Methods in Molecular Biology, vol. 140: Chaperonin Protocols
Edited by: C. Schneider © Humana Press Inc., Totowa, NJ
1
2
Quaite-Randall and Joachimiak
other chaperonins found in the archaea (for example,
7–9
, and others therein).
A sequence comparison of this chaperonin with others is given in
Fig. 1
.
We routinely purify this chaperonin by methods commonly used for other
chaperonins (
10
, and other chapters in this volume). The final yield of protein
can be enhanced by a slight heat shock to the culture (88°C for 30 min). Two
main properties, common to most chaperonins, are exploited during its purifi-
cation, that of its low isoelectric point (p
I
= 5.3) and size (_-subunit = 59.72
kDa; `-subunit = 59.68 kDa, and complex 9_ + 9` = 1074.6 kDa).
The chaperonin from the
Sulfolobus
species, with 18 subunits in the oligo-
mer, is the largest of the chaperonins studied to date. Gel-permeation chroma-
tography is therefore a very important step in the purification process. We use
Sephacryl high-resolution (HR) resins from Pharmacia (Uppsala, Sweden),
which are composite gels made by covalently crosslinking allyl dextran with
N
-
N,N
;-methylene
bis
-acrylamide to form a hydrophilic matrix of high-
mechanical strength an excellent flow properties.
Purification of the chaperonin from
S. shibatae
involves three chromato-
graphic steps, with concentration and dilution of the sample in between. A
detailed discussion of the chromatographic principles used can be found in
Chapter 3 of this volume. Detection of the chaperonin is either by native or
SDS-PAGE. The three chromatographic steps are:
1. Fast Q-Sepharose anion-exchange chromatography.
2. Gel-filtration chromatography on Sephacryl S-300 HR.
3. Mono Q-Sepharose HR anion-exchange chromatography.
These three steps can produce chaperonin, which is 98–99% pure on a mil-
ligram scale in 2–3 d. One interesting aspect of this chaperonin is that when
purified, it forms a doublet on a native gel. Initially we thought that this was
owing to an “empty” chaperonin ring oligomer and a larger, “loaded,” com-
plex between the chaperonin oligomer and bound substrate. This wass not
observed for GroEL or other Type I chaperonins. We thought that if it were
possible to purify each band, it might give some insight into the mechanism of
action of Type II chaperonins.
The two forms are separated by preparative continuous zone native gel
electrophoresis, in a preparative cell from Bio-Rad (Hercules, CA). This technique
is relatively simple and involves the use of gels of a uniform polyacrylamide con-
centration in conjunction with a single homogenous buffer system. In such a
system, sample is applied to the top of a gel, and separation occurs on the basis
of both the charge and the size properties of the proteins being analyzed. In this
case, electrophoresis conditions are optimized to maximize the differences be-
tween the two forms of the chaperonin from
S. shibatae
. During continuous
electrophoresis, protein leaving the bottom of the gel is washed with buffer and
Purification of Archaeal Chaperonin
3
Fig. 1. Sequence comparison of the S
ulfolobus shibatae
chaperonin (S-shi_55; S-
shi_56) with Tcp-1 from yeast (TCP_YEA) and GroEL from
E. coli
(GROEL).
collected in a fraction collector, in a manner similar to that used for column
chromatography. The two forms can then be studied individually. The high
solubility of the chaperonin at neutral pH is an important attribute during this
process, since concentration of the chaperonin in a tight band during electro-
phoresis does not cause precipitation. Sufficient chaperonin from each band
can be purified by this method for electron microscopy and circular dichroism
studies
(3)
. Since
S. shibatae
is a thermophilic archaea, all steps can be carried
out at room temperature. We have not tried to carry out the purification proce-
dure at lower temperatures. However because the chaperonin is stable during
4
Quaite-Randall and Joachimiak
storage at 4°C, it could be inferred that purification at this temperature would
not be detrimental to the protein.
2. Materials
Except where noted, all chemicals are purchased from Sigma (St. Louis,
MO) and are analytical grade or higher.
2.1. Cell Growth
S. shibatae
(DSM strain 5389) was obtained from Grogan et al.
(1)
. Cells
were obtained from J. Trent, who maintained the culture in liquid medium in a
150 L fermenter (
see
Note 1
).
1. Liquid culture medium:
a. 0.1% (w/v) Yeast extract
b. 0.1% (w/v) Sucrose
c. Salts: (Weight/L)
9.80 m
M
(NH
4
)
2
SO
4
1.29 g
2.00 m
M
KH
2
PO
4
272 mg
0.48 m
M
CaCl
2
52.8 mg
0.74 µ
M
FeCl
3
119 mg
d. Trace elements: (Weight/L)
7.5 µ
M
MnCl
2
0.93 mg
.75 µ
M
ZnSO
4
0.12 mg
.29 µ
M
CuCl
2
.038 mg
.12 µ
M
Na
2
MoO
2
.021 mg
e. pH should be adjusted to 3–5 with conc. H
2
SO
4
.
2.2. Chromatography Buffers
1. Lysis buffer: 50 m
M
Tris-HCl, pH 7.5, 0.5% Triton X-100 (v/v).
2. Buffer A: 50 m
M
Tris-HCl, pH 7.5 1 m
M
EDTA, 1 m
M
dithiotreitol (DTT), 50 m
M
NaCl.
3. Buffer B: 50 m
M
Tris-HCl, pH 7.5, 1 m
M
EDTA, 1 m
M
DTT, 1 M NaCl.
4. Buffer C: 50 m
M
Tris-HCl, pH 7.5, 1 m
M
EDTA, 1 m
M
DTT, 250 m
M
NaCl.
5. Buffer D: 50 m
M
Tris-HCl, pH 7.5, 1 m
M
EDTA, 1 m
M
DTT, 500 m
M
NaCl.
6. Buffer E: 50 m
M
Tris-HCl, pH 7.5, 1 m
M
EDTA, 5 m
M
DTT, 250 m
M
NaCl.
7. Buffer F: 50 m
M
Tris-HCl, pH 7.5, 1 m
M
EDTA, 5 m
M
DTT, 250 m
M
NaCl,
50% (v/v) glycerol.
2.3. Fast Q-Chromatography
Q-Sepharose Fast-Flow anion-exchange media are stored in 20% ethanol
and must be washed at least four times with 4 vol. of deionized H
2
O prior to
use. Media are suspended in 5 vol. of buffer A. A 30 × 2.3 cm column is
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Chaperonin Protocols (Methods in Molecular Biology) - Christine Schneider, medicalheaven radiology

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