Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-23
2002-10-22
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S235100, C435S320100, C435S069100, C435S069700, C435S243000, C536S023400, C536S023100
Reexamination Certificate
active
06468746
ABSTRACT:
FIELD OF THE INVENTION
The invention described herein relates to a method for selecting proteins which have high stability. Specifically, it relates to a system with which it is possible to screen out and to isolate the most stable proteins from a large number of mutants thereof.
BACKGROUND OF THE INVENTION
Proteins are widely used as enzymes or biocatalysts in industrial biotechnological processes. As antibodies, receptors, vaccines or hormones, they have a great potential for use in medical diagnosis and therapy. Unfortunately, however, there are limitations on use both in industry and in medicine. These derive in particular from the stability of the proteins being too low (Martinek, K. and Mozhanev, V. V., 1993). Biotechnological processes often proceed under reaction conditions which are survived for only a short time by the enzymes employed. Organic solvents, extreme pH values or high temperatures, which are advantageous or even necessary for many reactions, may lead to rapid inactivation of the enzymes (Gupta, S. and Gupta, M. N., 1993). Increased stability proves to be advantageous in the preparation of proteins, too. Thus, in general, the yields and the purity of a prepared protein can be improved if it has increased stability. An increased stability also extends the shelf life of proteins and simplifies storage and transport (Brems, D. N. et al., 1992). It is therefore of great interest to increase the stability of proteins. In the first place, a distinction must be made between different types of stability. Thermal stability (toward denaturation and aggregation), conformational stability (toward organic solvents) and chemical stability (toward oxidation, modification of the side groups) are particularly important on use as biocatalysts. Resistance to proteases is important in the medical sector. The thermodynamic stability is a measure of the equilibrium between folded, native (active) protein and its unfolded, denatured (inactive) form. An increase in the thermodynamic stability thus means a shift in the equilibrium toward native protein. The various types of stability correlate reasonably well. This means that a protein thermodynamically stabilized in its conformation has generally also undergone a stabilization according to the other criteria. This derives from the fact that irreversible inactivation (e.g. aggregation, degradation) mostly originates from unfolded protein, or inactivating alterations (e.g. chemical modification) can be averted better (Imoto, T., 1997).
There are in principle two possibilities for increasing the stability of a protein. On the one hand, proteins can be stabilized by external factors (e.g. solvent, immobilization, chemical modification) (Gray, C. J., 1993, Tyagi, R. and Gupta, M. N., 1993, Cabral, J. M. S. and Kennedy, J. F., 1993). On the other hand, the intrinsic stability of a protein can be increased by altering its amino acid sequence by mutations. This latter method is also referred to as protein engineering. Whereas the first method, external stabilization, rapidly reaches its limits through the conditions of use of the protein, the advantage of protein engineering is that it is possible thereby to stabilize proteins for applications under various conditions. It should be noted in this connection that the optimization of proteins in their physiological environment is not for thermodynamic stabilization but for folding rate, flexibility and degradability in the cell also (Shoichet, B. K. et al., 1995). It must therefore be assumed that there is sufficient potential available for stabilization by mutations.
In protein engineering in turn there are two ways of proceeding. On the one hand, stabilizing mutations can be deliberately introduced and, on the other hand, the stabilizing mutations can be selected out of a large number of randomly generated ones. Targeted mutagenesis demands extensive knowledge about the stabilizing interactions in a protein in order to have some probability of success. It is true that the principal types of interactions are known (e.g. Pace, C. N. et al., 1996), and computer-assisted algorithms moreover sometimes come quite close to predicting protein structures from the amino acid sequence (Fischer, D. and Eisenberg, D., 1996, Bowie, J. U. and Eisenberg, D., 1993). However, results of predicting the effect of individual mutations remain unsatisfactory. Even if the spatial structure of the protein to be modified is known, it is scarcely possible to predict or calculate the effects of mutations on protein stability because, in the end, knowledge about the denatured state of the protein or alternative conformations is still lacking. However, it is possible to apply targeted mutagenesis if homologs of the protein used are already known from thermophilic organisms. It is then possible, by sequence comparisons, to identify positions at which the protein may possibly be stabilized by directed mutagenesis.
Generation of a large number of randomly selected mutations with subsequent selection for the desired property is referred to as directed evolution. The advantage of directed evolution is that knowledge about the structure of a protein or about the interactions important for folding and stability is not a precondition. In line with Darwin's “survival of the fittest” there is cumulation of the mutants which come closest to the property which is being selected for. Two important preconditions must be met for application of directed evolution. In the first place, the property which is being selected for must in fact be selectable and, in the second place, it is necessary for the selected protein variants to be coupled with the nucleotide sequences coding for them. If it is possible to select for an activity necessary for growth of a microorganism (e.g antibiotic resistance), both conditions are met. Only those microorganisms which have developed this activity multiply. In order to achieve stabilization of proteins from mesophilic organisms, it is possible to incorporate them into a thermophilic organism and then to allow the latter to grow at appropriately elevated temperature. However, this method is applicable for only a few proteins because a precondition thereof is that the protein has an activity which is distinctly advantageous or even necessary for growth of the thermophilic organism. If the proteins to be stabilized do not have such an activity (which applies to most biocatalysts and, in particular, to proteins in medical therapy and diagnosis), it is necessary to carry out the selection indirectly or in vitro. Since in this case the proteins are separated from the organisms producing them there is initially the important problem of linkage to the nucleotide sequences encoding them. Various systems have been developed in the form of phage display (Smith, G. P., 1991, Patent WO 92/01047), cell surface display (Georgiou, G. et al., 1997), ribosome display (Mattheakis, L. C. et al., 1996, Hanes, J. and Plückthun, A., 1997), repressor display (Cull, M. G. et al., 1992) and selectively infectious phage display (SIP) (Krebber, C. et al., 1995, patent application EP 94102334) to make this coupling possible. However, application of these systems is essentially confined to the selection of binding properties, in particular that of single chain antibody fragments.
It is an object of the present invention to make the potential of directed evolution available for stabilizing proteins. Thus, the aim is to develop a method which makes it possible to select out of a large number of randomly generated mutants of any protein those having increased stability.
We have found that this object is achieved by the method presented here. It makes it possible for directed evolution to be used as method for stabilizing proteins. The selection criteria used for the thermodynamic stability is the resistance of a protein to proteolytic degradation, i.e. the stability to proteases. As described above, the various types of stability are closely interconnected. In particular, the correlation between protease resistance and thermody
Schmid Franz X.
Sieber Volker
BASF - Aktiengesellschaft
Guzo David
Keil & Weinkauf
Leffers, Jr. Gerald G.
LandOfFree
Method for selecting stabilized proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for selecting stabilized proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for selecting stabilized proteins will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2972360