Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-29
2002-01-29
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S094000, C436S501000, C536S025400
Reexamination Certificate
active
06342362
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to improvements in methods for the purification of macromolecules. In particular, it relates to methods for purifying macromolecules that have been added to a solution in order to carry out some desired purpose, but whose continuing presence is not desired. For example, a protease might be added during a step in the commercial production of an important product (e.g. blood factor VIII for hemophilia), but its removal might be necessary to carry out a subsequent step or to produce a sufficiently pure product for medicinal uses.
There are a large number of procedures that have traditionally been used to separate macromolecules, such as various chromatographic separations (by size, charge or affinity). These methods are generally time consuming, and appropriate methods vary with the target product and the identity of the contaminants. Proteins that have been genetically modified to have a run of histidines (a histidine tag) can be purified relatively simply on a nickel column (Hochuli et al.(1984),
J. Chromat
. 411:177; Porath (1992),
Protein Exp. Purif
. 2:263), and a number of companies (such as NOVAGEN, QIAGEN and InVitrogen) have designed systems for overexpressing such proteins and purifying them. These tagged proteins can also be attached to substrates for convenient manipulation, and can be repurified if desired. Proteins that have purified by other means, however, are less easily manipulated. For example, the enterokinase (a protease) cleavage site is often engineered into proteins in order to permit the removal of N-terminal sequences used in purification, but there is no straightforward procedure for quantitatively removing the enterokinase following the reaction. The absence of a general method for removing proteins (or other macromolecules) that have been added to a mixture puts a substantial constraint on the usefulness of many molecules.
SUMMARY OF THE INVENTION
This invention provides a means for removing a macromolecule (such as a protein molecule) from a mixture to which the purified macromolecule has been added. The macromolecule is modified by the addition of a tag following purification, and the tag can be used to remove the macromolecule once its purpose has been accomplished. In the preferred embodiment of this invention, a linking agent is used in which one portion of the linking agent comprises a tag that can be used for purification, while another portion of the linking agent comprises an active group that can bind to various parts of a macromolecule. For example, a linking agent can be made that consists of the maleimide group connected by a flexible carbon chain to six histidine residues. This linking agent can be used to attach the histidine tag to —SH or —NH
2
groups of macromolecules (such as the side chain of cysteine).
This invention also comprises the linking agents that can be used in the above procedure to repurify a macromolecule. In addition, kits are envisioned that contain materials for performing the above purification, such as a kit containing a linking agent, a compatible purification system, and additional reagents and instructions as necessary. For example, a kit might comprise the maleimide/his-tag linking agent described supra plus material for preparing a nickel column for purification. Alternatively, the nickel column might be replaced by a syringe containing a similar purification matrix, so the mixture containing the maleimide/his-tag modified protein and other components can be purified by pushing it through the syringe.
In some situations, it might be necessary to purify the modified protein away from the unreacted linking agent in order to prevent the linking agent from reacting with other components of the mixture to which the modified protein is to be added. This may be accomplished by a size separation or by using a relatively low ratio of linking agent to target and purifying the target protein using the tag. These two procedures could be combined such that a target is first reacted with an excess of linking agent and separated from the unreacted linking agent by size, and the target population is then purified using the tag to ensure that all molecules are tagged. Batches that have been prepared in this fashion can in turn form the nucleus of kits. For example, a kit might comprise a protease that has been tagged in this fashion plus a purification system and appropriate additional reagents and instructions.
DEFINITIONS
Terms herein generally follow their normal, accepted meanings in the art, and may be found in any of the common text books or laboratory manuals, such as
Biochemistry
(Lehninger, Worth Publishers, 1975),
Biochemistry
(Stryer, W.H.Freeman and Company, 1988),
Molecular Cloning: A Laboratory Manual
(Sambrook et al., Cold Spring Harbor Press, 1989),
Current Protocols in Molecular Biology
(Ausubel et al., Wiley Interscience, 1994) and
Chemistry of Protein Conjugation and Cross-Linking
(Wong, CRC Press, 1991).
The term “linking agent”, as used herein, refers to a non-naturally occurring molecule comprising a tag and a binding region. In some unusual implementations, these regions could overlap.
The term “tag”, as used herein, refers to a molecule that can be attached to a larger macromolecule, and which can be used to separate that macromolecule from macromolecules that do not have the tag.
The term “binding region”, as used herein, refers to the region of a linking agent that can attach the linking agent to specific regions of macromolecules under appropriate conditions. The attachment will generally be covalent, but need not be, though it must be strong enough so that there will not be a substantial amount of separation during purification.
DETAILED DESCRIPTION
A. Designing Appropriate Linking Agents
Linking agents are created by combining appropriate tags and binding regions. Tags and binding regions are well known to those of ordinary skill in the art, though they have never been used for the sorts of purposes envisioned herein. While the applications described herein use known tags and binding regions as exemplars, there is no reason why any newly discovered tag or binding region would not be comparably appropriate.
Tags are typically used in the isolation of proteins. For this purpose, the DNA that encodes the tag is usually attached to one of the ends of the corresponding cloned gene, which causes the expressed gene to produce a tagged protein product suitable for isolation. Commonly used tags include runs of histidines (“his-tag”), portions of glutathione S-transferase (“GST-tag”) and a variety of short sequences that can be recognized by antibodies. Some such tags are described in catalogs from laboratory suppliers, such as NOVAGEN, InVitrogen and QIAGEN. Important features of a tag include its ability to facilitate purification (such as by binding tightly to an appropriate column or antibody), its ability to fold reasonably independently, and its lack of interference with the function of the tagged protein. Since the tags envisioned herein will attach to a variety of positions on a protein, it is likely that many of these attachments will have little effect on the function of the protein, which is a substantial improvement over the conventional method in which a tagged protein is produced from a cloned gene, with the tag being present in a specific position that might well cause problems. Methods for purifying proteins that have one of these tags attached are well known to those of ordinary skill in the art.
Binding regions are well known in the literature concerned with creating bifunctional molecules that will act as cross-linking agents. For example, Wong provides a wealth of information on designing and selecting appropriate molecules (see Wong,
Chemistry of Protein Conjugation and Cross-Linking
, CRC Press, 1991 and references therein). An appropriate choice is typically a binding region that reacts relatively non-specifically with the target molecule, but does not react with any component of itself (a binding region that reac
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