Purified protein G from streptococcal bacteria

A process is disclosed for recovering a cell wall protein, particularly a Fc-receptor, from streptococcal bacteria. In the process, streptococcal bacteria are treated with at least one proteolytic enzyme to solubilize the cell wall protein. A process is also disclosed for recovering Fc-receptor type III (protein G) having selective IgG-binding capability (i.e., no albumin-binding capability) from streptococcal bacteria by pretreating these bacteria with enzymes prior to said enzymatic solubilization.

BACKGROUND OF THE INVENTION 
This invention relates to a process for recovering a cell wall protein from 
streptococcal bacteria. More particularly, the invention relates to a 
process for recovering a Fc-receptor from such bacteria. 
Fc-receptors from bacteria and processes for recovering these are known and 
described in the literature. For example, U.S. Pat. No. 3,850,798 
describes a process for recovering the Fc-receptor protein A from 
staphylococcal bacteria through enzymatic solubilization using the enzyme 
trypsin. 
A similar process using fag-induced cell wall-decomposing enzyme has also 
been suggested for solubilization of Fc-reactive protein from group A 
streptococci. This latter process constitutes one example of the principle 
that, by decomposing the long sugar polymers and the cross-linking 
peptide-bridges in the cell wall, a solubilization of all substances which 
were bonded to this structure will result. Residues of the cell wall may 
be left on the solubilized molecules. Simultaneously with the 
solubilization, all soluble intracellular substances from the 
streptococcal bacteria are released in the obtained liquid, which 
therefore will be very complex. 
The former process, which according to U.S. Pat. No. 3,850,798 can be used 
in connection with staphylococcal bacteria, is a more selective process. 
However, such process according to our knowledge has neither been used nor 
described in connection with streptococcal bacteria. On the contrary, such 
use with streptococcal bacteria should have been deemed to be 
non-operative in view of the results which presently are known with 
respect to the protein nature of Fc-receptors and the sensitivity of these 
surface structures to proteolytic treatment. As a rule, a complete 
degradation of the proteins to amino acids and peptides are obtained, and 
all biological activity is lost. 
SUMMARY OF THE INVENTION 
It has now been surprisingly found in accordance with the present invention 
that streptococcal bacteria can be treated with proteolytic enzyme to 
solubilize cell wall protein. In particular, it has been found by the 
present invention that solubilized Fc-receptor type III (designated below 
as protein G) can be recovered from such treated bacteria. 
Fc-receptors such as protein A and protein G have useful characteristics, 
for example the ability to bind to the Fc-portion of immunoglobulins. 
Fc-receptors can thus be advantageously be used as well as therapeutic as 
in analytical connections. One example of such a useful application is 
extra-corporeal treatment of blood in connection with some autoimmune 
diseases, wherein the Fc-receptor could be used as an agent to remove 
so-called immune complexes from the blood. In comparison to protein A, 
protein G has however several advantages which make protein G far 
superior. For example, protein G can bind to all IgG-subclasses, while 
protein A cannot bind to human IgG III. Furthermore, protein G lacks the 
ability to bind IgA and IgM. Thus, protein G is in a sense a more 
selective Fc-receptor than protein A, which binds also to these 
immunoglobulin classes. 
According to our present knowledge, no simple process exists for recovering 
Fc-receptors, particularly protein G, from streptococcal bacteria. The 
need to find such a process therefore is great. 
DETAILED DESCRIPTION OF INVENTION 
The present invention provides a process for recovering a cell wall protein 
from streptococcal bacteria. The streptococcal bacteria are treated with a 
proteolytic enzyme to solubilize the cell wall protein. The solubilized 
cell wall protein is preferably isolated from the treatment suspension. 
Suitable proteolytic enzymes that can be employed in the process of the 
invention include papain, trypsin and/or pepsin. Papain is a preferred 
enzyme. 
As already mentioned, the recovered Fc-receptor from streptococcal bacteria 
can bind to all IgG-classes. Experiments have shown that the Fc-receptor 
(protein G) also bind to albumin which is a useful protein in blood 
plasma. This means that a competition between IgG and albumin to the 
binding surface on protein G thus can exist, when protein G is used in 
connection with an extracorporeal treatment of blood to remove immune 
complexes, as previously mentioned. 
According to the invention, it has also been found that a still more 
selective protein G, i.e. a protein G which binds only to IgG but not to 
albumin, can be obtained from streptococcal bacteria. Such more selective 
protein G is obtained from bacteria which are exposed to an enzymatic 
pretreatment prior to the solubilization. This enzymatic pretreatment also 
preferably employs proteolytic enzymes. Preferred pretreatment enzymes 
include trypsin and/or pepsin. 
The solubilized cell wall protein can be isolated and recovered in the way 
as described in the European Patent Publication No. 0 046 915. Briefly, in 
this known method the solubilized protein, possibly after filtration of 
bigger impurities, is brought into contact with a ligand having affinity 
to said protein to form a complex. The ligand is immobilized on a soluble 
carrier. The complex is then purified from possible similar impurities and 
thereafter split to release the protein. The released protein is then 
separated and recovered through filtration. For further details of this 
known method, reference is made to said European patent publication, the 
disclosure of which is incorporated herein in by reference. 
A similar method, which can also be used according to the present 
invention, is described in U.S. patent application Ser. No. 07/129,935, 
filed Dec. 3, 1987, now U.S. Pat. No. 4,783,264, in the names of Nylen et 
al., the disclosure of which is incorporated herein by reference. 
An effective amount of the enzyme is employed to solubilize the desired 
protein. Also, the enzymes employed in the process of the invention are 
preferably used in the form of a suspension. Preferably, the enzyme 
suspensions contain between about 50 and about 250 .mu.g., preferably from 
about 75 to about 150 .mu.g., enzyme per ml of a 10% bacteria suspension.

The following examples are presented for purposes of demonstrating, but not 
limiting, the process of the invention. 
EXAMPLE 1 
Human group G streptococcal bacteria, G 148, cultured in Tood/Hewitt-broth, 
were suspended (10% suspension) in 0.01M tris-HCl, pH 8.0. One hundred 
.mu.l 0.4M L-cystein and 10 .mu.l papain of varying concentration in the 
same buffer were added per ml bacterial suspension. The mixture was 
incubated for 1 hour at 37.degree. C. Jodoacetamide was added to provide a 
final concentration of 6 mM and a bacterial suspension was obtained. 
The so obtained bacterial suspension was centrifugated (2000 g) for 30 
minutes. Thereafter, the supernatant was ultracentrifugated (50000 g), 
frozen immediately at -80.degree. C., and used as starting material for 
isolation of the protein through sequential use of ion exchange 
chromatography on DEAE-cellulose gel, gel filtration on Sephadex G-100 and 
affinity chromatography on Sepharose 4B-bonded IgG. 
The isolated protein migrated as a homogeneous band when analysed on 
SDS-PAGE. Also, on agarose gel electrophoreses only one band was seen in 
the alfa.sub.1 -region, indicating a high degree of purity. 
SDS-PAGE in disks and in rods (labeled protein G) both gave an apparent 
molecular weight of 30500. Treatment of the protein with 2-mercaptoethanol 
did not influence the result. 
EXAMPLE 2 
0.5 mg pepsin is added per ml of 10% bacterial suspension (streptococcal 
bacteria of the same kind as in Example 1 above) in 0.1M acetate buffer, 
ph 4.0.mu. and was incubated for 30 minutes at 37.degree. C. Tests 
according to known methods on the remaining bacterial bodies showed that 
they completely lacked the ability to bind albumin, while the ability to 
bind IgG was maintained unaffected. 
These pepsin-pretreated bacteria were washed in 0.1M tris-HCl, pH 8.0, and 
diluted to a 10% suspension in this buffer. Thereafter, the enzmatic 
solubilization with papain was performed in the same way as described 
above in Example 1 to recover a protein G having the ability to 
selectively bind IgG. 
It will be understood that the embodiments described herein are merely 
exemplary and that a person skilled in the art may make many variations 
and modifications without departing from the spirit and scope of the 
invention. All such modifications and variations are intended to be 
included within the scope of the invention as defined in the appended 
claims.