Method of purification of carboxypeptidase G.sub.1

A method of purifying a protein-reaction mixture to obtain carboxypeptidase G.sub.1, which method comprises: passing the mixture through a column containing support material, such as carboxymethyl cellulose, under pH conditions such that the carboxypeptidase G.sub.1 is preferentially and selectively bonded to active sites on the support material in preference to other protein materials of the mixture; displacing the bound carboxypeptidase G.sub.1 by an eluant, such as a glutamate, which has a greater affinity for the carboxypeptidase G.sub.1 than the support material; and recovering from the column a mixture of the eluant and the carboxypeptidase G.sub.1.

BACKGROUND OF THE INVENTION 
Folate antagonists, particularly methotrexate, have become important drugs 
in the treatment of certain human malignancies. Methotrexate 
(4-amino-N.sup.10 -methylpteroylglutamic acid) is chemically similar to 
folic acid and its derivatives (particularly 5-methyltetrahydrofolate in 
human tissue) and interferes with folic acid and derivatives thereof to 
interrupt folate metabolism in the synthesis of DNA in the body. A 
specific example is the inhibition of enzyme dihydrofolate reductase 
caused by methotrexate. This process inhibits folate metabolism and 
thereby the synthesis of DNA. 
The primary use of the enzyme is for the depletion of serum folates. A 
secondary use is in methotrexate rescue. Where methotrexate has worked in 
the body for an optimum time, it is necessary to stop its work in human 
tissue, since death is caused by too high a concentration of methotrexate 
in the human body for too long a period of time. 
An enzyme, carboxypeptidase G.sub.1 (hereinafter designated as "G.sub.1 "), 
has been isolated which hydrolyzes the carboxyl-terminated glutamate from 
both reduced and nonreduced folate derivatives. The enzyme, G.sub.1, 
specifically breaks down the methotrexate and serum folates which then 
become inactive. 
Attempts to purify the reaction mix and to obtain the particular 
carboxypeptidase G.sub.1 by conventional techniques have proved 
inadequate, probably due to the similar chemical structure and molecular 
weight of other protein materials in the crude reaction mixture. There is 
a need then to provide a simple, rapid and inexpensive method of 
separating carboxypeptidase G.sub.1 from the preparative reaction mix. 
A method of preparing G.sub.1 containing a reaction mixture is described in 
the article "Purification and Properties of Carboxypeptidase G.sub.1," by 
J. L. McCullough et al, The Journal of Biological Science, Vol. 246, No. 
23, pages 7207-7213, herein incorporated by reference. 
SUMMARY OF THE INVENTION 
Our invention relates to a method for the purification and/or concentration 
of a protein from a mixture of proteins, such as a crude reaction mixture. 
In particular, our invention concerns the purification of enzymes like 
carboxypeptidase, such as G.sub.1, from a crude reaction mixture 
containing the enzyme and to the product produced by the method. 
We have discussed that a selected protein from a mixture of proteins, and 
optionally other products as might be present in a crude reaction mixture 
produced by a microorganism, may be concentrated and purified in a rapid, 
simple and inexpensive technique. Our method comprises: preferentially and 
selectively binding the selected enzyme protein to a support material 
containing sites which complex with or bind the protein molecule; 
displacing the bound protein with an eluant which is selected to have a 
higher affinity for the bound protein than the support material; and 
recovering the eluant and selected protein mixture. The protein is then 
recovered by conventional recovering methods from the eluant protein 
mixture. 
In one embodiment, the enzyme, carboxypeptidase G.sub.1, is purified from a 
crude reaction mixture containing the G.sub.1, such as a reaction mixture 
obtained by the method in the McCullough et al publication, supra. The 
crude reaction mixture contains a mixture of proteins from which it is 
most difficult to separate the G.sub.1 by other methods. Our method for 
purifying the G.sub.1 enzyme includes passing the crude reaction mixture 
through a column containing gel or particulate support material with 
active carboxyl groups under selected pH conditions, such that the G.sub.1 
is selectively complexed or bound to the active sites of the support 
material in preference to the other components in the mixture. Our method 
then includes displacing the bound G.sub.1 by passing an eluant, such as a 
carboxyamino acid or acid salt, through the column, the carboxyamino 
material having a greater affinity and binding or complexing degree than 
the G.sub.1 to the support material. Our method then includes recovering 
the carboxyamino-G.sub.1 mixture and recovering the G.sub.1 from such 
mixture. 
The protein mixture from which the selected protein is to be recovered or 
purified may be derived from a variety of natural and synthetic sources, 
such as pharmaceutical waste and by-products, pure protein mixtures, crude 
reaction mixtures of protein derived from microorganism reaction and 
cultures and the like. 
The support material may be placed in contact with the protein mixture by 
any method of contact; however, typically and preferentially, the support 
material is placed in a column as a packing, and contact is achieved by 
passing the protein mixture through the column, for example, by gravity or 
under pressure. The support material is usually in finely divided particle 
or gel form which permits the passage of the liquid protein mixture in 
solution or suspended form. 
The nature of the support material may vary, provided only that it contains 
active sites or otherwise complexes and binds only or preferentially the 
selected protein under the pH conditions of the method. Typically, the 
support material would comprise a compound which has a plurality of acidic 
groups or sites, such as carboxy groups. One class of materials found to 
be useful as support materials is carboxylated cellulosic materials, such 
as carboxymethyl cellulose. Other materials would include, but not be 
limited to, carboxyethyl and carboxypropyl; e.g., alkyl, cellulose and 
mixtures and combinations thereof, as well as various synthetic polymers, 
such as used in ion-exchange columns like acrylate resins with free acid 
groups, of which bind the amino carboxy groups of the selected protein. 
In the preferred and optimum embodiment, the pH of the mixture in the 
column is adjusted and maintained at a selected pH or pH range, so that 
the protein is weakly bound to the support material. The optimum pH is 
easily determined, and, for example, with G.sub.1 is about 6.0, with 
carboxymethyl cellulose as a support material. At a pH of less than about 
6.0, the G.sub.1 is unstable, while at a pH of above 6.0, the protein 
G.sub.1 is not fully charged and, therefore, exhibits less affinity for 
the support material. If desired, other factors, such as the amino-acid 
concentration, may be adjusted to regulate the optimum binding pH of the 
protein. In the purification of G.sub.1, the method is carried out with a 
solution which contains a zinc ion; e.g., 1 .times. 10.sup.-4 molar, since 
G.sub.1 is unstable in the absence of zinc ion. 
The eluant solution used to displace the bound protein is selected to be a 
compound, typically an acidic compound, preferably an amino acid, which 
has a greater degree of affinity for the protein than the support 
material, so that the protein is displaced and from which the displaced 
protein may be easily or rapidly removed. The eluant is usually a 
pH-buffered solution and, for example, also contains a zinc-ion 
concentration in the purification of G.sub.1. The eluant material 
preferentially is an amino acid or an amino salt, such as glutamic acid, 
or the soluble salts, such as sodium or potassium glutamate, and similar 
low-molecular weight amino acids. Such amino acids would comprise amino 
alkane polycarboxylic acids and salts; for example, amino C.sub.3 -C.sub.6 
alkane di or tri carboxylic acids and their salts. Often, a review of the 
literature or the publication covering the support material will provide 
degrees of affinity for various amino acids for the support material and 
for the protein, so that a selection of the desired eluant can be made or 
the choices narrowed to a few materials to be tested. Glutamic acid or 
sodium glutamate is a desired eluant for G.sub.1, although other 
carboxyamino acids and acid salts may be employed. The eluant solution is 
passed through the column by gravity or under pressure, with the column 
worked one, two, three or more times until all the bound protein is 
removed, and the eluant-protein solution mixture recovered. 
The protein is then separated from the eluant, such as, for example, by gel 
filtration, reverse osmosis, dialysis, fractionation, electrophoresis, or 
other separation and purification methods, to permit the recovery of the 
protein and the reuse, if desired, of the eluant in the method. 
In operation, a chromatographic column is packed with the support material 
and the support material is equilibrated with a buffer solution to the 
desired pH, and with a metal ion or other material, if required, as a 
stabilizer for the protein. The protein mixture is then poured into the 
column and the column worked several times with a buffer solution until 
all the unbound protein of the protein mixture is washed from the column. 
The buffered, stabilized eluant solution is then passed through the column 
several times, and the resulting mixture is recovered and the protein 
removed from this eluant-protein mixture. 
Our method will be described for the purpose of illustration only in 
connection with the purification of carboxypeptidase G.sub.1 from a 
reaction mixture; however, it is recognized and is within the spirit and 
scope of our invention that other proteins may be purified from other 
protein mixtures by the same or a similar method, and by changes and 
modifications to our method within the skill of the art.

DESCRIPTION OF THE EMBODIMENTS 
A crude reaction mixture of protein containing carboxypeptidase G.sub.1 was 
prepared as set forth in the McCullough et al publication, supra. 
Reaction cells (9Kg) were suspended in two volumes (1 g/l) of tris - Zn 
buffer (0.01 M tris Cl - 10.sup.-5 M ZnCl.sub.2, pH 7.3) and passed 
through a laboratory homogenizer four times. Three additional cell volumes 
(27 l) of tris - Zn buffer were added to the suspension, and the pH 
adjusted to 7.3 with 1 N NaOH. Gross cell debris was removed in a 
centrifuge (14,000 .times. g, 20 l/hr), and the supernatant clarified in 
an ultracentrifuge with an RK 6 rotor (80,000 .times. g, 17 l/hr). 
A one percent solution of protamine sulfate (22 l) was added to the 
clarified extract (39 l) in a ratio of 1.0g of protamine sulfate per 3 g 
of protein. The resulting suspension was stirred for 45 minutes and 
centrifuged in a centrifuge as above. The precipitate, which contained the 
bulk of the nucleic acids, was discarded. 
The protamine sulfate supernatant (58 l) was brought to 55% saturation with 
solid ammonium sulfate (321 g/l). The suspension was stirred for 30 
minutes and centrifuged batchwise for 10 minutes. The precipitate was 
discarded and the supernatant (66 l) brought to 80% saturation with solid 
ammonium sulfate (22.4 lbs). The suspension was stirred for 30 minutes, 
and centrifuged in an ultracentrifuge with an RK 3 rotor (40,000 .times. 
g, 20 l/hr). The supernatant was discarded and the precipitate dissolved 
in approximately its volume of tris - zinc buffer. The solution was 
desalted on a 7 l G-25 column (10 .times. 100) (two runs 700 ml/run). The 
precipitate which formed during desalting was removed by centrifugation 
(10,000 .times. g, 30 min.). 
The clarified sample (3.8 l) was mixed with a swollen cake of QAE Sephadex 
A50 (50 g dry weight) which was equilibrated with tri - Zn buffer. The 
suspension was stirred for 30 minutes and filtered on a buchner funnel. 
The filter cake, which was not allowed to dry, was subsequently washed 
with tris - Zn buffer (4000 ml). The initial unabsorbed solution and the 
buffer wash were pooled, and the filter cake discarded. 
The unabsorbed protein solution (7.9 liter) was brought to 85% saturation 
with solid ammonium sulfate (559 g/l). The suspension was stirred for 30 
minutes and centrifuged in an ultracentrifuge (40,000 .times. g, 20 l/hr). 
the supernatant was discarded and the precipitate was dissolved in a 
minimal volume of a succinate - Zn buffer (0.005 M Na succinate - 
10.sup.-4 M ZnCl.sub.2, pH 6.0). The solution (775 ml) was desalted on the 
G-25 column as above (387 ml sample per run). The precipitate which formed 
during desalting was removed by centrifugation (10,000 .times. g, 30 
minutes). The supernatant (approximately 3000 ml) was stored frozen. 
The desalted frozen supernatant sample was thawed and applied to a (14 
.times. 50) CM column packed by single decantation with microgranular 
carboxymethyl cellulose equilibrated with 0.005 M Na succinate - 10.sup.-4 
M ZnCl.sub.2 (pH 6.0) buffer solution. The support material was CMC 52 
from Whatman Limited. The column was washed with one column volume of 
starting buffer solution, and then extensively washed (7 - 10 column 
volumes) with 0.007 M Na succinate - 10.sup.-4 M ZnCl.sub.2 (pH 6.0) to 
remove all unbound protein from the column, with the carboxypeptidase 
G.sub.1 then bound to the carboxyl-active sites of the CMC support 
material. Determination of the removal of unbound protein may be made by 
measuring the optical density (OD) at 280 m.mu. of the column wash 
effluent with a spectrophotometer. Washing is carried out until the OD at 
280 m.mu. is less than 0.010 OD. 
The bound carboxypeptidase G.sub.1 was then eluted with a 0.015 M sodium 
glutamate - 10.sup.-4 M ZnCl.sub.2 (pH 6.0) solution, the amino acid 
glutamate having a greater affinity for the G.sub.1 than the G.sub.1 has 
for the CMC, thereby displacing the G.sub.1 from the CMC column support 
material. The eluant flow rate throughout the operation of the column was 
approximately 2.5 l per hour. Fractions of 800 ml volume were collected 
during the loading and washing procedures, while fractions of 
approximately 220 ml were collected during the elution of the enzyme. The 
peak fractions containing homogeneous carboxypeptidase G.sub.1 were pooled 
and stored frozen for later separation of the G.sub.1 by gel filtration. 
The drawing graphically depicts the purification process for 
carboxypeptidase G.sub.1. Absorbence (in this particular case 280 m.mu.) 
is plotted against the volumes of the various liquids passing through the 
chromatographic column during the purification. The dotted line represents 
the change in absorbence as the impure protein mixture is first loaded on 
the carboxymethyl cellulose in the column and then eluted with sodium 
glutamate to displace the carboxypeptidase G.sub.1 fraction, and finally 
washed with a sodium-chloride solution to remove other proteins from the 
carboxymethyl cellulose. The solid line quantitatively represents the 
amount of purified carboxypeptidase G.sub.1. 
Table I contains data from specific purifications of carboxypeptidase 
G.sub.1 using carboxymethyl cellulose. Activity is represented in units 
and unit percentage, and is determined by the methods explained in the 
publication, supra. 
TABLE I 
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Affinity Elution of Carboxypeptidase G.sub.1 on 
Carboxymethyl Cellulose 
RUN A RUN B* 
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Column dimensions, cm 
14 .times. 50 
7 .times. 20 
Column volume, liters 
7.7 0.8 
Flow rate, liters/hour 
2.5 0.5 
Fractions collected (milli- 
liters), load and wash 
1600 200 
Fractions collected (milli- 
liters), enzyme elute 
220 22 
Activity loaded, units.sup.1) 
5.7 .times. 10.sup.5 
1.2 .times. 10.sup.5 
Activity recovered, percent 
95 102 
Activity recovered by affinity 
elution, percent 61 95 
Specific activity loaded, 
units per milligram of protein 
16.0 16.0 
Specific activity recovered by 
affinity, units per milligram 
of protein elutes 670.0 770.0 
Purification, fold 42 48 
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*Average of two identical runs 
.sup.1) units = micromoles of substrate hydrolyzed per minute