Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1999-05-26
2000-06-13
Carlson, Karen Cochrane
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
435 691, 435333, 435334, 435335, 435344, 530402, 530413, 530417, A23J 100, C07K 100
Patent
active
060751274
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a modification method for facilitating the preparation of purified (poly)peptides and to a purification process using this modification method.
2. Description of the Related Art
During the last years solid phase peptide synthesis, using either t-Boc or F-moc strategies, has been largely improved. Sophisticated protocols of synthesis allowed the preparation of polypeptides of about 100 residues or more.sup.1-4. Nevertheless, incomplete coupling and chain termination that may occur at any cycle of the peptide assembly leads to the formation of deletion--and truncated sequences.
This and the possible occurrence of side reactions observed mainly during the final cleavage from the resin hamper the straightforward isolation of the desired peptide from other impurities. The purification of long synthetic polypeptides is a major problem in the production of products useful for biological studies and for human and animal use where a high level of purity is mandatory.
In particular, when sequences containing 30 or more residues are synthesized, the differences in physical properties such as size, charge and hydrophobicity between the desired product and deleted, truncated or modified peptide impurities may be too small to allow adequate purification. In addition, the modern powerful separation techniques, i.e. reverse phase HPLC, are often limited by low yields and small sample loadability which is time-consuming and expensive. Different approaches have been already tested to circumvent this limitation. Biotinylation of a 153 residue IL-1 synthetic protein.sup.5 and of a 99 residue SIV protease synthetic protein.sup.6 were performed and the biotinylated chains were isolated on an avidin-agarose column.
Ball et al..sup.7 have recently proposed a purification procedure based on the addition of a reversible protecting group which bears either lipophilic, acidic or basic functions to the last residue of the peptide chain.
More specific chromatographic methods have been optimized exploiting the presence of particular residues in the synthesized sequences. For example, cysteine-containing peptides have been purified by reaction with immobilized mercury derivatives.sup.8 or activated thiols.sup.9 and immobilized metal ion affinity chromatography (IMAC).sup.10 has been successfully applied for the purification of peptides containing histidine or tryptophan.sup.11.
In recombinant proteins a histidine tail, B cell epitope or GST moieties were purposely added. These tails could subsequently be used in affinity chromatography.
In general, a purification protocol which is based on the physico-chemical properties of the synthesized peptide has to be optimized for each individual sample, which is a time-consuming and costly exercise. For these reasons, a number of techniques have been developed to render purification procedures of general applicability.sup.15. However, the methods described so far are not completely satisfactory since they are still time consuming and/or leave covalently derivatized peptides in the final purified products which may pose some concern for their biological and physico-chemical properties and their final utilization in animals and humans.
It is therefore the object of the present invention to provide an improvement to the known methods by providing a method for the modification of (poly)peptides for facilitating purification thereof, and to a purification process for (poly)peptides, which method and process are universally applicable, result in a high yield of recovery and are easy to perform.
This object was achieved according to the invention by a modification method, which comprises the insertion of at least one specifically cleavable amino acid at the end of the (poly)peptide chain during synthesis thereof and protecting the same amino acid(s) within the (poly)peptide, if present, against cleavage, in order to allow for specific cleavage precisely at the specifically cleavable amino acid(s).
SUMMARY OF THE IN
REFERENCES:
Hochuli et al. Genetic Appropach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology 6(11): 1321-1325, 1988.
Sassenfeld, Helmut, M. Engineering Proteins for Purification. Trends in Biotechnology. 8(4) 88-93, 1990.
Krieger et al. Affinity Purification of Synthetic Peptides. Proc. Natl. Acad. Sci. 73(9): 3160-3164, 1976.
Corradin Giampietro
Fasel Nicolas
Reymond Christophe
Roggero Mario
Carlson Karen Cochrane
RMF Dictagene SA
Schnizer Holly
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