Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1991-09-26
1994-09-06
Wityshyn, Michael G.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
530395, 530413, 530416, C07K 1506
Patent
active
053449150
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel proteins and the preparation thereof.
TNF.alpha. (tumor necrosis factor) is a known protein which has a broad spectrum of biological activities. It influences various malignant and non-malignant cell types, plays a part in septic shock and tissue injuries and in kidney rejections, transplantations, shock lung and cerebral malaria (Lymphokines 1987 Vol. 14; Pharmaceutical Res. 5 (1988), 129; Science 234 (1986), 470; Nature 330 (1987), 662; J. Exp. Med. 166 (1987), 1132; Science 237 (1987), 1210; J. Exp. Med. 166 (1987), 1280).
It is known that the action of TNF.alpha. can be neutralized by antibodies (EP 260 610). However, these antibodies are not human substances so that use on humans may lead to immunological reactions.
We have now found proteins which are of human origin and are able to neutralize the action of TNF.alpha..
The present invention relates to proteins which have a molecular weight of about 42,000 daltons and have at the N terminus the amino acid sequences Xaa Thr Pro Tyr Ala Pro Glu Pro Gly Set Thr Cys Arg Leu Arg Glu where Xaa is hydrogen, a phenylalanine residue (Phe) or the amino acid sequences Ala Phe, Val Ala Phe, Gln Val Ala Phe, Ala Gln Val Ala Phe, Pro Ala Gln Val Ala Phe or Leu Pro Ala Gln Val Ala Phe, and the muteins thereof.
By muteins are meant proteins which are produced by suitable exchange, deletion or addition of amino acids or peptides in the protein chain without this leading to a large reduction in the action of the novel proteins. Muteins can also be obtained by altering the glycoside residue.
The novel proteins described herein have acidic properties, their leoelectric point being at pH 2 to 5. They bind very specifically to TNF.alpha. and are digestible by trypsin with difficulty or not at all.
The novel proteins can be isolated, for example, from the urine of patients with fever, ie. whose body temperature is about 38.degree. C. or above. For this purpose, the urine is first concentrated, which can be effected, for example, by reverse osmosis or ultrafiltration. The retentate from this is then purified by ion exchange and affinity chromatography.
The proteins can also be obtained from ascites fluid from human patients with ovarian carcinomas.
The proteins can be purified by conventional methods such as affinity or ion exchange chromatography.
The proteins obtained in this way are inhomogeneous in the amino acid sequence at the N terminus. Up to 7 amino acids may be absent. Inhomogeneities of this type are not unusual with endogenous proteins and also occur, for example, in .gamma.-interferon.
Treatment with an endoglycosidase alters the migration behavior of the protein in SDS polyacrylamide gel electrophoresis, and this is attributable to elimination of sugar residues.
The proteins described herein are present in urine and ascites fluid in concentrations of from 1 to 100 .mu.g/l. Known genetic engineering methods (cf. Maniatis, T. et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1982) can be used to obtain the protein in larger Mounts for pharmaceutical purposes. It is necessary for this purpose initially to identify the genetic information for the novel protein and to isolate the corresponding nucleic acid. This entails the pure protein being reduced with dithiothreitol, then iodacetamide is added to derivatize the free SH groups, and subsequently the protein which has been treated in this way is cleaved with cyanogen bromide and then with trypsin into small peptides. The peptides are fractionated by reverse phase chromatography. N-terminal sequencing of one of these purified peptides reveals the sequence Val Phe Cys Thr Lys. The protein also contains the following three peptide sequences: Gly Val Tyr Thr Set, Ile Cys Thr Cys Arg Pro Gly Tyr and Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Xab Thr Thr Ser Ser Asp Ile Cys Arg Pro, where Xab is an amino acid which has yet to be identified and is possibly glycosylated.
The available peptide sequences now permit, by synth
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Daum Lothar
Doerper Thomas
Hillen Heinz
LeMaire Hans-Georg
Moeller Achim
BASF - Aktiengesellschaft
Sayala C.
Wityshyn Michael G.
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