Process for the reactivation of denatured protein

The invention concerns a process for the reactivation of denatured protein, in which the protein is incubated with a solution of Tris base or/and a salt of Tris at a concentration of at least 400 mmol/l and at a pH at which the protein to be treated can take up its native conformation.

CROSS REFERENCE TO RELATED APPLICATIONS 
This application is a continuation of PCT/EP91/02190, filed Nov. 21, 1991. 
DESCRIPTION 
The present invention concerns an improved process for the solubilization 
and renaturation of denatured protein wherein the protein is treated with 
a Tris buffer which has a concentration of at least 400 mmol/l of Tris 
base or of a Tris salt. 
When proteins are produced in prokaryotic cells such as E. coli, 
difficultly soluble proteins aggregates (inclusion bodies) are often 
formed. Solubilization and renaturation steps are necessary in order to 
convert these proteins into their active form. The solubilization of 
proteins is a known process (see e.g. EP 0 361 475 A1, EP-A 0 114 506, 
EP-A 0 093 619 and EP-A 0 253 823). 
In addition buffers as well as processes for the renaturation of 
denaturated proteins are known (see e.g. WO 87/02674, EP-A 0 364 926, EP 0 
241 022). 
An important factor in the reactivation of proteins (with or without 
disulphide bridges) which limits the yield of renatured protein is the 
competition between conversion of the denatured protein into the correct 
folding intermediate and the aggregation of several protein molecules. For 
this reason the concentration of denatured protein in the renaturation 
buffer is an important parameter for the yield of the renaturation process 
i.e. increasing concentrations of denatured protein promote aggregation 
and lower the relative yield of renatured protein with the conformation of 
the native protein. 
In all processes known at present for the reactivation of proteins it is 
therefore necessary that the amount of denatured protein in the reaction 
mixture does not exceed a critical concentration. Since the protein is 
often only sparingly soluble in the reactivation buffer used, this 
therefore results in considerable disadvantages regarding lower yield, 
large amount of time needed and/or larger buffer volumes. 
An object of the present invention is therefore to provide conditions for 
the reactivation of denatured proteins (i.e. in particular for the 
solubilization and renaturation), and to provide a buffer by which means 
the solubility of the denatured and renatured protein is substantially 
increased in comparison to known buffers. 
This object is achieved according to the present invention by a process for 
the reactivation of denatured protein which is characterized in that the 
protein is incubated with a solution of Tris(hydroxymethyl)aminomethane 
base (denoted Tris in the following) disclosure or a salt of Tris at a 
concentration of at least 400 mmol/l and at a pH value at which the 
protein to be treated can take up its native conformation. 
The use of a buffer which contains Tris base or salts of Tris at a 
concentration of more than 400 mmol/l for the reactivation of denatured 
proteins considerably increases the solubility of the renaturing proteins. 
This leads to a substantial increase in the yield of active protein in 
comparison to known standard methods. Although previously known 
reactivation buffers often contain Tris at a concentration of 50 to 100 
mmol/l in order to buffer the reaction solution, the surprising property 
to mediate solubilization (and thus the ability to improve the 
renaturation yield) of Tris at a concentration of at least 400 mmol/l has 
previously not been recognized. 
A salt of Tris within the meaning of the present invention is understood as 
a Tris salt of an arbitrary organic or inorganic acid. Examples of Tris 
salts are for instance Tris acetate, Tris benzoate, Tris borate, Tris 
carbonate, Tris citrate, Tris-HCl, Tris maleate, Tris nitrate, Tris 
oxalate, Tris phosphate, Tris succinate, Tris sulphate and suchlike. 
The process according to the present invention is suitable for the general 
renaturation of proteins whereby the cause of the denaturation (salt, heat 
etc.) is not in fact critical. Although the process according to the 
present invention is preferably suitable for the renaturation of products 
produced by genetic engineering and which occur in an inactive form as 
inclusion body material, the process can, however, in principle also be 
applied to other denatured proteins. In particular the process according 
to the present invention is applicable to the processes disclosed in WO 
87/02673, EP-A 0 241 022 and EP-A 0 364 926. In this connection WO 
87/02673 discloses a process for the activation of non-glycosylated tPA 
after expression in prokaryotes by cell lysis, solubilization under 
denaturing and reducing conditions and reactivation under oxidizing 
conditions in the presence of GSH/GSSG, in which a pH value of 9 to 12, a 
GSH concentration of 0.1 to 20 mmol/l, a GSSG concentration of 0.01 to 3 
mmol/l and a non-denaturing concentration of a denaturing agent are used 
in the reactivation step. EP-A 0 241 022 discloses a process for the 
renaturation of denatured proteins in solution in a renaturation buffer in 
which a solution of the protein to be renatured is prepared at the 
critical concentration in the chosen buffer and, after formation of the 
folding intermediate, further protein to be renatured is added in an 
amount necessary to achieve the critical concentration. EP-A 0 364 926 
concerns a process for the activation of biologically active proteins 
produced by genetic engineering and expressed in prokaryotes after cell 
lysis by solubilization under denaturing and reducing conditions and 
subsequent reactivation under oxidizing and renaturing conditions in which 
a protein concentration of 1-1000 .mu.g/ml is used and dialysis is carried 
out between solubilization and reactivation against a buffer which has a 
pH value between 1 and 4, and contains 4 to 8 mol/l guanidine 
hydrochloride or 6 to 10 mol/l urea. 
The process according to the present invention can be carried out in one of 
two ways. One variant is to work with a Tris buffer of the stated 
concentration so that Tris or/and a Tris salt is also used for adjusting 
the pH. The second variant is to work with a buffer which has previously 
been described for the corresponding process and to additionally add Tris 
or/and a salt of Tris. This means that the pH value of the incubation 
solution is adjusted by a buffer substance which is different from Tris. 
In both cases it is expedient to take care that the addition of Tris or 
the increase in Tris concentration does not result in a change in pH. 
The incubation is carried out at a pH value at which the protein to be 
treated can exist in a native conformation. This means that the incubation 
in the process according to the present invention does not take place at a 
pH value which does not allow the formation of a native protein 
conformation. Native protein conformations are in turn understood to 
include such secondary, tertiary and, if desired, quarternary structures 
in which the protein can have biological activity. 
The process according to the present invention comprises the incubation of 
a denatured protein with a Tris solution which has a concentration of at 
least 400 mmol/l. The Tris concentration is preferably 0.4 to 2 mol/l, 
particularly preferably about 1 mol/l Tris. With some proteins (e.g. with 
antibody fragments) optimal reactivation yields are already achieved at 
Tris concentrations in the range of 0.5 mol/l. 
The yield of a renaturation process depends, as already described above, on 
the concentration of the protein in the renaturation solution. For the 
process according to the present invention a protein concentration is 
preferably chosen which is in the range up to 4000 .mu.g/ml. Depending on 
the type of protein and the renaturation process, protein concentrations 
exceeding this range can, however, also prove to be suitable. 
In the process according to the present invention the denatured protein is 
either added continuously or batchwise (e.g. pulse renaturation) to the 
renaturation solution. In many cases (e.g. in the renaturation of tPA and 
tPA derivatives) it has also proven to be advantageous to add 0.2 to 1 
mol/l arginine to the incubation solution. 
Preferred examples of proteins which can be renatured by the process 
according to the present invention are recombinant tPA or tPA muteins (in 
particular a non-glycosylated tPA mutein having the domain composition 
K2P), recombinant granulocyte colony stimulating factor (G-CSF) or 
antibodies or their fragments. The process according to the present 
invention is, however, not limited to these examples but rather can be 
applied to any protein. 
The advantages of the reactivation process according to the present 
invention include, in particular, an increase in the final yield of active 
protein of 30 to 300% compared to a process in which a buffer is used 
which has a lower Tris concentration. Moreover, the concentration of 
denatured protein in the renaturation buffer can be increased without 
losses in the final yield i.e. the renaturation process becomes 
substantially quicker and more effective. 
In particular the process according to the present invention is 
advantageous for pulse renaturation in a renaturation reactor. In this 
case the yield of renatured protein is increased and in addition the 
protein concentration per pulse can be increased, which results in a 
shortening of the reactivation period. It is also possible to pulse up to 
a higher final protein concentration in the renaturation preparation 
without observing a reduction in the renaturation yield. As a consequence 
the buffer volume is considerably reduced. A pulse renaturation procedure 
that has proven to be particularly advantageous is one in which the 
renaturation is carried out in a buffer containing arginine and a Tris 
concentration of about 1 mol/l where the concentration of the protein to 
be renatured is increased by about 200 .mu.g/ml per pulse. 
It is intended to further elucidate the invention by the following 
examples. 
______________________________________ 
Abbreviations: 
______________________________________ 
GSH reduced glutathione 
GSSG oxidized glutathione 
tPA tissue plasminogen activator 
C.sub.prot protein concentration 
Arg arginine 
Gdn guanidine 
renat. renaturation 
CK creatine kinase 
______________________________________