Solubilization of protein after bacterial expression using sarkosyl

This invention relates to two methods for solubilizing proteins which are rendered insoluble by bacterial expression. One method comprises directly lysing the host bacterial cells with the detergent Sarkosyl. The other method comprises conventional lysing of the bacteria followed by an extraction process using Sarkosyl and fractionation. These methods render the proteins soluble. They do not entail harsh denaturation of the proteins, and therefore do not require renaturation of the proteins in many cases. Rather, they render the proteins soluble, in their native form.

FIELD OF THE INVENTION 
This invention relates to two methods for solubilizing bacterially 
expressed proteins which are rendered insoluble by the bacterial 
expression process. These methods utilize the detergent Sarkosyl (N lauryl 
sarcosine or any other detergent which is an N amide derivative of 
sarcosinate). One method is a direct lysis of host bacterial cells in the 
presence of Sarkosyl detergent. The other method is an extraction process 
performed on insoluble protein, also requiring Sarkosyl detergent. 
During purification of bacterially expressed proteins, proteins may become 
insoluble due to a variety of mechanisms. One mechanism which renders 
proteins insoluble is co-aggregation of the proteins with bacterial outer 
membrane components, such as outer membrane proteins and 
lipopolysaccharides. Once the proteins become insoluble, strong 
denaturants are necessary for resolubilization. The methods of this 
invention allow for the solubilization of bacterially-expressed proteins 
which are rendered insoluble by the bacterial expression process without 
resorting to strong denaturants, such that functional protein may be 
recovered and purified. In particular, the methods of this invention allow 
for the efficient separation of solubilized bacterially-expressed proteins 
from contaminating outer membrane components, with subsequent removal of 
the detergent. 
BACKGROUND OF THE INVENTION 
Bacterial expression systems have been utilized to produce large quantities 
of homogeneous proteins whose function may then be assayed and studied 
irrespective of the sequence of that protein or its abundance in its 
natural host. However, the bacterial expression process renders many 
proteins insoluble. Once a protein is rendered insoluble, the protein must 
be solubilized. 
Certain methods have been developed to solubilize proteins which have been 
rendered insoluble by bacterial expression. However, the solubilization 
methods created heretofore necessarily entail denaturation of the protein. 
As a result, the protein must necessarily be renatured. This renaturation 
process may be costly and time consuming. In many instances, renaturation 
is not possible. Therefore, a need has arisen to create a method for 
solubilizing bacterially-expressed proteins which does not denature the 
proteins, and therefore eliminates the necessity to renature altogether. 
SUMMARY OF THE INVENTION 
This invention relates to two methods for solubilizing 
bacterially-expressed proteins, wherein the proteins are not denatured, 
but rather may be separated from contaminating bacterial outer membrane 
components while remaining in their native form. These nondenatured 
proteins may then be assayed for their function and purified without the 
necessity of renaturing the proteins prior to purifying and assaying the 
proteins. 
The proteins to be studied are first expressed in bacterial systems, which 
requires inserting the coding sequence for a protein in an appropriate 
plasmid expression vector. During the lysis of the bacteria, the proteins 
are rendered insoluble. The first method of this invention comprises 
solubilizing-bacterially expressed proteins by directly lysing the 
bacteria with the detergent Sarkosyl. The second method comprises 
conventional lysing methods (such as the French press method, sonication, 
or lysozyme digestion followed by freezing and thawing) followed by the 
extraction of insoluble protein with Sarkosyl and EDTA, followed by 
fractionation.

DETAILED DESCRIPTION OF THE INVENTION 
Bacterial expression is a versatile process which allows for the production 
of any protein for commercial or research purposes. However, a problem 
which arises with some proteins during bacterial expression is that the 
proteins can only be extracted in an insoluble form. Furthermore, strong 
denaturants have heretofore been necessary for their subsequent 
solubilization. This invention allows for the solubilization of insoluble 
proteins after bacterial expression. The methods of this invention, in 
contrast with other methods of solubilizing proteins, do not require 
denaturation and subsequent renaturation of the protein. This is a 
particular advantage for the classes of protein which cannot be renatured. 
The methods of this invention therefore allow for commercial use of 
bacterial expression for many proteins heretofore not suitable for 
bacterial expression because it yields soluble, nondenatured protein. 
This invention comprises two procedures for lysing the bacteria and 
fractionating the lysates, consisting of: (1) lysing the bacteria by a 
light lysozyme digestion followed by the addition of Sarkosyl detergent, 
and several obligate steps of fractionation following lysis, and (2) 
lysing the bacteria by other standard methods, collecting the insoluble 
protein and extracting this insoluble protein with Sarkosyl detergent and 
EDTA, followed by fractionation. 
A protein which may be solubilized after bacterial expression by the 
methods of this invention is actin in either its full-length (42-kDa) form 
or in a truncated (29-kDa) mutant form. Actin is normally a soluble 
protein. Its insolubility after bacterial expression and lysis may be due 
to co-aggregation of the actin with bacterial outer membranes during 
bacterial lysis. This method can also be used on other proteins, including 
Dictyostelium discoideum myosin light chain kinase. Sarkosyl detergent is 
a relatively mild chaotrope with a specific effect upon this co 
aggregation process since it does not solubilize the bacterial outer 
membrane. The Sarkosyl lysing method of this invention produces soluble 
actin after bacterial expression. 
EXAMPLE 1 
Solubilization of Bacterially-Expressed Actin by Direct Sarkosyl Lysis 
Culture Growth 
Plasmid expression vectors were constructed as described in Frankel et al., 
"Expression of Actin in Escherichia Coli", Journal of Biological 
Chemistry, Vol. 265, No. 29, Oct. 15, 1990. For expression of actin in 
large-scale culture, a 1/100 dilution of an overnight culture was 
inoculated into 1 liter of M10+medium (60 mM Na.sub.2 HPO.sub.4, 11 mM 
K.sub.2 PO.sub.4, 37 mM NH.sub.4 Cl, 9.0 mM NaCl, 1.0 mM MgSO.sub.4, 0.05 
mM CaCl.sub.2, 0.05 mM MnCl.sub.2, 0.03 mM FeCl.sub.3, 0.5% casamino 
acids, 1% glycerol, 5% LB broth, made with autoclaved distilled H.sub.2 O 
followed by filter sterilization) plus 40 ug/ml ampicillin, incubated at 
37.degree. C. in a 2 liter baffled flask with vigorous shaking, grown to 
an OD.sub.550 of 0.2-0.3 and then induced with 4 mM IPTG. When the cells 
reached an OD.sub.550 of 0.95, the flasks were chilled in an ice-water 
bath for 20-30 min. Enriched minimal medium is used because growth in 
strict minimal medium is very slow. After the cultures were grown, 
Sarkosyl lysis was performed. 
Direct Sarkosyl Lysis 
All procedures were at 4.degree. C. or on ice. Bacteria from one liter of 
culture grown to an absorbance at 550 nm of 0.95 were sedimented and 
washed with a buffer containing 20 mM Tris, pH 8 at 4.degree. C., and 50 
mM NaCl. Washed cells were resuspended in 15 mls of STE (10% sucrose, 100 
mM Tris, pH 8 at 4.degree. C. 1.5 mM EDTA), and lysozyme was added to 100 
.mu.g/ml to perform a light digestion. Lysozyme digestion may be 
facilitated by a buffer containing EDTA, sucrose and Tris (pH 8.0). The 
cells were incubated on ice 10-15 min. or until lysis competent. Lysis 
competence may be assessed by resuspending a small volume of bacterial 
suspension into a 200 fold excess of distilled water (hypotonic solution). 
The cells were then added to 132 mls of divalent cation free dilution 
solution with a pH of 8.0, which is capable of maintaining the tonic 
balance of the bacteria and the stability of the expressed protein. This 
dilution solution may comprise 50 mM NaCl, 15 mM buffer at pH 8.0, 0.5-5 
mM dithiothreitol, and protease inhibitors. After dilution, the volume may 
be between 5 and 15 times the volume of the cell suspension during 
lysozyme digestion. To lyse the bacteria, while stirring, 3 mls of 10% 
Sarkosyl was added to a 0.2% final concentration. For lysis, the Sarkosyl 
may be added to a final concentration of 0.2-2%. While adding Sarkosyl, 
the rate of stirring was increased to compensate for the increased 
viscosity, but turbulence was avoided. After the addition of Sarkosyl, the 
lysate contained: 15 mM triethanolamine (pH 8 at 4.degree. C.), 50 mM 
NaCl, 2.5 mM ATP, 1.0 mM GDP, 1.0 mM DTT, 20 ug/ml aprotinin, 10 ug/ml 
leupeptin, 5 ug/ml pepstatin, 2.5 ug/ml chymostatin, 0.43 mM PMSF, 0.43 mM 
o-phenanthroline, 10 mM Tris, 0.16 mM EDTA, 1.0% sucrose (the last three 
were from STE). GDP was included to maintain EF Tu in a native state. 
After 2 min., the lysate was mildly sonicated to reduce viscosity due to 
high molecular weight nucleic acid: seven 10 second bursts at 90 watts 
(Heat Systems-Ultrasonics, setting 2). The lysate was then centrifuged at 
32,000.times.g for 11 min. so as to separate bulk soluble protein from 
insoluble material. The supernatant was collected and the Sarkosyl was 
sequestered by adding octylglucoside to a concentration of 2%, using a 25% 
stock. After stirring the supernatant for 5 min., MgCl.sub.2 and 
CaCl.sub.2 were added to concentrations of 1.25 mM and 1.06 mM 
respectively. The estimated free concentration of each divalent cation was 
0.1 mM. After stirring for 20 min. this fraction was centrifuged at 
60,000.times.g for 12 h to pellet 30 S ribosome subunits. 
In general, Sarkosyl may be sequestered by adding nonionic detergent in at 
least a 5-fold weight excess over the Sarkosyl detergent. This nonionic 
detergent may be octylglucoside. Sarkosyl sequestration may be followed by 
the addition of divalent cations to stabilize the protein. 
Sequestration may also be followed by one of three methods to remove all 
detergent. One method, applicable to sequestration using any nonionic 
detergent, involves binding the protein to a solid resin matrix and 
washing all of the detergent out of the resin matrix. A second method, 
applicable only to sequestration using octylglucoside, involves dialysis 
in which the dialysis membrane has a molecular weight cut-off of at least 
30,000 daltons. A third method, applicable only to sequestration using 
octylglucoside, involves concentration of the sample using a 30,000 dalton 
cut off membrane followed by dilution of the concentrated sample into a 
detergent-free solution. Another method of removing detergent does not 
require sequestration. This alternative method involves precipitation of 
Sarkosyl by adding a several millimolar excess of divalent cation over 
EDTA and removing the Sarkosyl by centrifugation. 
RESULTS 
1 Actin Is Soluble After Direct Sarkosyl Lysis 
After French press lysis, bacterially expressed actin is insoluble. FIG. 1 
represents fractionation of both 29-kDa actin and 42-kDa actin after 
French press lysis. The fractions were analyzed by immunoblots, and the 
immunoblots were quantitated by densitometry. However, soluble full length 
42-kDa actin and soluble truncated 29-kDa actin were recovered after 
direct Sarkosyl lysis. Lane 1 is a sample of total lysate; lane 2 is a 
sample of the supernatant after low speed centrifugation; lane 3 is a 
sample from the pellet after low speed centrifugation. The relative 
loadings of lanes 1 and 2 are equal, while 4 times more sample was loaded 
in lane 3. The lane labelled MW contains molecular weight markers. 
Table I shows fractionation of actin after Sarkosyl lysis. FIG. 2 
represents a Coomassie stained gel and an immunoblot of the fractions 
listed in Table I. Panel A is a Coomassie stained gel and Panel B is an 
immunoblot of the same samples. Lane 1 is a sample of total lysate; lane 2 
is a sample of the low speed supernatant; lane 3 is a sample from the low 
speed pellet; lane 4 is a sample of postribosomal supernatant; lane 5 is a 
sample from the ribosome pellet. Each lane is loaded so as to have roughly 
equal amounts of 29-kDa actin, as determined by Western analysis. 
TABLE I 
______________________________________ 
SOLUBILITY OF ACTIN AFTER SARKOSYL LYSIS 
AND OCTYLGLUCOSIDE SEQUESTRATION 
______________________________________ 
29 kDa Actin 
Total Lysate 100% 
Low Speed Supernatant 59% 
Low Speed Pellet (Outer Membrane) 
37% 
Post-Ribosomal Supernatant 
19% 
Ribosome Pellet 18% 
42 kDa Actin 
Total Lysate 100% 
Low Speed Supernatant 93% 
Low Speed Pellet (Outer Membrane) 
6% 
Post-Ribosomal Supernatant 
55% 
Ribosome Pellet 17% 
______________________________________ 
Lysis and fractionation were performed. Yields were quantitated by the 
densitometric scanning of immunoblots. In adition, it was determined that 
the low and high speed supernatants contained approximately 160 and 75 mg 
of DNA, respectively. The high speed centrifugation was overnight; some 
loss of actin occurred at this step, probably due to proteolysis. While 
these numbers are from one experiment, comparable results have been 
obtained in repetitions of the experiment. 
2. 42-kDa Actin Binds DNase I During Purification 
A characteristic property of actin is the ability to bind DNase I. In the 
course of purifying 42-kDa actin, it was possible to demonstrate its 
ability to bind DNase I. The complete purification was performed as 
follows: 
A low speed supernatant was obtained as described under "Sarkosyl lysis," 
octylglucoside and divalent cations were added, sodium azide was added to 
0.02%, and the solution was batch absorbed to DNase 1 affinity resin for 
2.5 hours. The resin was batch washed with 15 bed volumes of high salt 
buffer (25 mM triethanolamine, pH 8.0 at 4.degree. C., 0.8M NaCl, 0.02% 
azide, 1.0 mM ATP, 1.0 mM GDP, 5.0 mM NaPP, 0.25 mM DTT, 0.1 mM EDTA, 1.38 
mM McCl.sub.2, 0.945 mM CaCl.sub.2, 0.5 mM PMSF; the estimated free 
concentration of each divalent cation was 0.1 mM). The resin was then 
loaded into a column and washed with 5 bed volumes of high salt buffer. 
Most of the buffer above the bed was drained, and 100% deionized formamide 
equal to 30% of the bed volume was added, gently resuspending the resin 
and incubating for 10 min. The resin bed was completely drained, and 
residual liquid was removed by pushing air through the bed. 40% formamide 
disrupts the interaction of actin with DNase I, and allows actin to be 
eluted from the affinity resin. The eluate was diluted by the addition of 
high salt buffer, so that the formamide concentration was 30%. The eluate 
was clarified of resin fines, and immediately loaded onto a Sephadex 
G-150column (1.5.times.68 cm) equilibrated in a modified high salt buffer 
(25 mM triethanolamine, 0.8M NaCl, 0.02% azide, 0.1 mM EDTA, 5 mM NaPP, 
0.2 mM ATP, 0.5 mM DTT, 0.138 mM CaCl.sub.2 ; free CA.sup.++ was 
estimated at 5 uM). The column was run using the modified high salt 
buffer, and monomeric 42-kDa actin was pooled based upon a previous 
calibration of the column with rabbit actin. The pool was concentrated by 
vacuum dialysis against G-buffer (2.5 mM triethanolamine, pH 8.0, 0.5 mM 
DTT, 0.2 mM ATP, 0.1 mM CaCl.sub.2, 0.01% sodium azide), to a volume of 
250 .mu.l. Polymerization was initiated by adding ATP to 1 mM, MgCl.sub.2 
to 4 mM, NaCl to 50 mM, and phalloidin to 25 .mu.M. After an overnight 
incubation at 0.degree. C., the F-actin sample was dialyzed for 6 hrs 
against Actin EM Buffer (2 mM Pipes, pH 6.8 at 4.degree. C., 1 mM ATP, 4 
mM MgCl.sub.2, 50 mM NaCl, 0.5 mM DTT, and 0.02% sodium azide). After 
dialysis, phalloidin was added to 25 .mu.M, and the F-actin was stored on 
ice until used for negative stain electron microscopy. 
FIG. 4 represents the purification of 42-kDa actin, and also illustrates 
its binding to DNase I affinity resin. Panel A is a Coomassie stained gel 
at three stages in the purification. Lane 1 is a sample of the low speed 
supernatant, which was the fraction absorbed to DNase I affinity resin; 
lane 2 is a sample of the eluate from the DNase I affinity resin, which 
was the fraction loaded onto the gel filtration column; lane 3 is a sample 
of the final 42-kDa actin sample, pooled from the gel filtration column, 
concentrated and polymerized. Panel B is an immunoblot of fractions from 
the gel filtration column. Lane 1 is a sample from a fraction in the void 
volume; lane 2 is a sample from the pool of fractions containing monomeric 
actin. 29-kDa actin elutes in the void volume of the column, with some co 
migrating 42-kDa actin. The majority of the 42-kDa actin elutes as a 
discrete peak of monomers. 
3. Purified 42-kDa Actin Binds Mycsin S-1 in an ATP Sensitive Manner 
The two most characteristic properties of native actin are the ability to 
polymerize into filaments and the ability to bind myosin. After 
purification, the actin exhibits both properties. FIG. 5 shows filaments 
of polymerized actin solubilized by this procedure are able to bind myosin 
S-1 in an ATP sensitive manner. Since the denaturation of actin is 
irreversible, these properties indicate that the actin was never denatured 
by Sarkosyl lysis. Panel A shows actin filaments incubated with myosin S-1 
in the presence of ATP. Electron microscope grids were negatively stained 
with 1% uranyl acetate. The magnification of Panels A and B are the same. 
The inset shows actin filaments from a sample which was not incubated with 
myosin. The magnification bar represents 26.7 nm. Panel B shows actin 
filaments incubated with myosin S-1 in the absence of ATP. Grids were 
stained as in Panel A. The distance between consecutive myosin S-1 
arrowheads is 35.5 nm.+-.0.11 (S.E.). The magnification bar represents 40 
nm. 
4. 29-kDa Actin Binds DNase I 
Most forms of actin bind to DNase I with very high affinity. It was 
therefore possible to assay a relatively crude bacterial fraction from the 
actin producing strain using DNase I affinity chromatography. 
All procedures for the DNase I Binding Assay were at 4.degree. C. or on 
ice. The high speed supernatant obtained after Sarkosyl lysis was 
supplemented with PMSF to 0.5 mM and sodium azide to 0.02%. The following 
were added to tubes containing DNase I resin or control resin: 10 ml 
portions of the supernatant and either a DNase I stock solution or stock 
buffer. The reaction mixtures were gently mixed for 2 h, the resins 
pelleted, the unbound fractions removed, and the resins resuspended in 4 
bed volumes of high salt buffer (25 mM triethanolamine pH 8 at 4.degree. 
C., 800 mM NaCl, 0.02% sodium azide, 1.0 mM ATP, 1.0 mM GDP, 5 mM NaPP, 
0.5 mM DTT, 0.1 mM EDTA, 1.38 mM MgCl.sub.2, 0.95 mM CaCl.sub.2, 0.5 mM 
PMSF: the estimated free concentration of each divalent cation was 0.1 
mM). The resins were gently mixed with high salt buffer for 30 min., 
pelleted, and the washes removed. In most experiments the wash was 
repeated a second time and both high salt washes were added to the initial 
unbound fraction. One ml of this combined flow-through+high salt wash 
fraction was ethanol precipitated, and solubilized for gel analysis. 
The binding of D.d. actin was measured by adding pure actin to control 
lysates and manipulating these lysates in the same manner as lysates which 
contained 29-kDa actin. The binding of 29-kDa and D.d. actin to DNase I 
was quantified by immunoblots. Table II shows 29-kDa actin s ability to 
bind DNase I was equivalant to that of D.d. actin. As a control, the 
binding of both types of actin to mock resin was tested, and none was 
found to bind. Another control involved binding to affinity resin in the 
presence of soluble DNase I. 
TABLE II 
______________________________________ 
BINDING OF 29-kDa AND D.d. ACTIN TO DNase I 
PERCENT BINDING* 
______________________________________ 
29 kDa Actin 
CONTROL-SEPHAROSE (without 
-1% .+-. 2% (4)** 
covalently attached DNase I) 
DNase I-SEPHAROSE 20% .+-. 2% (6) 
DNase I-SEPHAROSE + 6% .+-. 2% (2) 
0.5 mg of FREE DNase I 
DNase I SEPHAROSE + -1% .+-. 2% (4) 
2.0 mg OF FREE DNase I 
D.d. ACTIN 
CONTROL-SEPHAROSE (without 
3% (1) 
covalently attached DNase I) 
DNase I-SEPHAROSE 19% .+-. 3% (2) 
DNase I-SEPHAROSE + N.D.*** 
0.5 mg of FREE DNase I 
DNase I-SEPHAROSE + 0% .+-. 2% (2) 
2.0 mg OF FREE DNase I 
______________________________________ 
*Percent of the loaded fraction retained by the column 
**Values are presented as the mean .+-. SE, when n &gt; 2; the number of 
independent binding reactions is indicated in parentheses 
***Not determined 
A high speed supernatant was obtained as in Table I. Portions of this were 
batch absorbed to DNase I affinity resin. The resin used in each binding 
reaction contained 0.6 mg of immobilized DNase I. After separating the 
unbound fraction, the resin was washed twice with high salt buffer. The 
washes were combined with the unbound fraction, and the amount of actin 
present in this fraction was quantitated by immunoblots. The binding of 
D.d. actin was measured by adding pure actin to control lysates, and 
manipulating these lysates in the same manner as lysates which contain 29 
kDa actin. 
EXAMPLE 2 
Solubilization of Bacterially Expressed Actin Obtained After French Press 
Lysis by the Sarkosyl Extraction Method 
Culture Growth 
Culture growth was performed the same as in Example 1. 
French Press Lysis+Sarkosyl Extraction 
At this point all procedures were done at 4.degree. C. or on ice. The 
bacteria were washed with low salt buffer (10 mM triethanolamine, pH 8 at 
4.degree. C., 0.5 mM ATP, 0.5 mM DTT, 0.1 mM CaCl.sub.2) and resuspended 
to a final volume of 20 mls of low salt buffer+protease inhibitors (20 
ug/ml aprotinin, 10 ug/ml leupeptin, 2.5 ug/ml pepstatin, 2.5 ug/ml 
chymostatin). Lysis was in the French pressure cell at 1000 lb/in.sup.2., 
running the lysate through twice. The lysate was centrifuged at 
116,500.times.g for 8 min. (the equivalent of 15,000.times.g for one hour) 
to collect insoluble protein. Then, Sarkosyl extraction was performed as 
follows: the supernatant was removed and the pellet resuspended in 10 ml 
of a divalent cation free extraction buffer (1.5% Sarkosyl, 25 mM 
triethanolamine, pH 8.0, 4 mM ATP, 0.8 mM dithiothreitol, 1 mM EDTA, 0.02% 
sodium azide, 20 ug/ml aprotinin, 5 ug/ml leupeptin, 2.5 ug/ml pepstatin, 
2.5 ug/ml chymostatin and 0.5 mM o-phenanthroline) with 30 strokes of a 
Teflon-glass homogenizer. The effective ranges in the extraction buffer 
are as follows: Sarkosyl 0.5-2%, pH 6.0-9.0, and 0.5-5 mM EDTA. The 
extraction buffer may contain any other constituents necessary for the 
stability of the expressed protein. The resuspension was then 
re-centrifuged at least at the rate performed before Sarkosyl extraction, 
here at 116500.times.g for 16 min., to separate the solubilized protein 
from the insoluble material. 9 ml of the supernatant was immediately added 
to 58.5 ml of octylglucoside (OG) buffer (to sequester Sarkosyl in 
micelles of octylglucoside). The final concentrations after dilution were 
2.0% octylglucoside, 0.2% Sarkosyl, 25 mM triethanolamine, 0.8M NaCl, 1.0 
mM ATP, 0.2 mM dithiothreitol, 0.02% azide, 0.13 mM EDTA, 20 .mu.g/ml 
aprotinin, 5 .mu.g/ml leupeptin, 2.5 .mu.g/ml pepstatin, 2.5 .mu.g/ml 
chymostatin, 0.07 mM o-phenanthroline. After this was mixed, divalent 
cations were added (0.68 mM CaCl.sub.2 gave a free concentration of 0.1 
mM); high speed centrifugation was then performed. 
In general, Sarkosyl may be sequestered by adding nonionic detergent in at 
least a 5-fold weight excess over the Sarkosyl detergent. This nonionic 
detergent may be octylglucoside. Sarkosyl sequestration may be followed by 
the addition of divalent cations to stabilize the protein. 
Sequestration may also be followed by one of three methods to remove all 
detergent. One method, applicable to sequestration using any nonionic 
detergent, involves binding the protein to a solid resin matrix and 
washing all of the detergent out of the resin matrix. A second method, 
applicable only to sequestration using octylglucoside, involves dialysis 
in which the dialysis membrane has a molecular weight cut-off of at least 
30,000 daltons. A third method, applicable only to sequestration using 
octylglucoside, involves concentration of the sample using a 30,000 dalton 
cut off membrane followed by dilution of the concentrated sample into a 
detergent free solution. Another method of removing detergent does not 
require sequestration. This alternative method involves precipitation of 
Sarkosyl by adding a several millimolar excess of divalent cation over 
EDTA and removing the Sarkosyl by centrifugation. 
RESULTS 
1. Actin is Soluble After French Press Method+Sarkosyl Extraction 
After French press lysis, bacterially-expressed actin is insoluble. FIG. 3 
represents fractionation of both 29-kDa actin and 42-kDa actin after 
French press lysis and Sarkosyl extraction. FIG. 3 shows that insoluble 
actin recovered after French press lysis was solubilized by Sarkosyl 
extraction. However, the bacterial outer membrane proteins were not 
solubilized by Sarkosyl extraction, and could be completely separated from 
the solubilized actin. Panel A is a Coomassie-stained gel. Panel B is an 
immunoblot of the same samples as in Panel A. Lane 1 is a sample of total 
lysate; lane 2 is a sample from the low speed pellet; lane 3 is a sample 
of the Sarkosyl extraction supernatant; lane 4 is a sample from the 
Sarkosyl extraction pellet. 
2. 29-kDa Actin Binds DNase I After Sarkosyl Extraction 
The high speed supernatant obtained after Sarkosyl extraction was bound to 
DNase I affinity resin. The ability of 29-kDa actin to bind was equivalent 
to that seen in Table II after Sarkosyl lysis. 
EXAMPLE 3 
Solubilization of Myosin Light Chain Kinase, Obtained After French Press 
Lysis, by the Sarkosyl Extraction Method 
Dictyostelium discoidium myosin light chain kinase is not a structural 
protein, like actin, but is an enzyme which specifically phosphorylates 
the myosin light chain. When expressed in E. coli, the kinase is 
insoluble, and the aggregates can only be solubilized using strong 
denaturants such as 8M urea. However, tests performed at Stanford 
University show that the kinase can be differentially extracted from the 
aggregates with Sarkosyl detergent (0.5%+1 mM EDTA), such that virtually 
all of the kinase is solubilized but bacterial proteins (presumably outer 
membrane proteins) are not. The extracted kinase is still enzymatically 
active. Starting from the same quantity of aggregated protein, 10-fold 
more kinase activity is recovered using Sarkosyl solubilization when 
compared to urea solubilization. Soluble kinase is also obtained when 
bacteria are lysed in the presence of Sarkosyl (Sarkosyl lysis), but the 
kinase obtained by this procedure was not extensively characterized for 
the retention of enzymatic activity. 
The results obtained with the kinase are significant for the following 
reasons: (1) the protein is significantly different from actin, (2) the 
protein has an easily quantitated enzymatic activity, (3) the protein is 
expressed to very high levels in E. coli, unlike full length wild type 
actin, and is therefore more representative of commercial applications of 
this technology.