DE3931839A1 - NEW PROTEINS AND THEIR PRODUCTION - Google Patents

Abstract

A new protein is described which has a molecular weight of about 15,000 daltons and the N-termination SDYEFPPPKKXRPG. The protein is suitable for the control of disease and the protecion of grazing cattle from ticks.

Description

The present invention relates to new proteins and their production.

It is already known that leeches inhibit blood clotting Produce substance, the hirudin (see The Merck Index, 10th edition, No. 4613). Hirudin is a polypeptide with a molecular weight of approximately 6500.

Substances that inhibit blood clotting also get blood from other people sucking parasites, e.g. the ticks. These substances are but so far neither in pure form nor synthetically produced (Natural Sciences 11, 30 (1961).

A new protein from ticks has now been isolated.

The invention relates to a new protein which is a molecular has a weight of about 15,000 daltons and the amino at the amino terminus acid sequence

SDYEFPPPKKXRPG

in which X is S or N, and its muteins.

Muteins are proteins that differ from the new protein by exchange, deletion and / or addition of amino acids or peptides arise in the protein chain without the effect of the new Proteins changed significantly.

The new protein can be isolated from ticks. For this the ticks in a buffer at pH 6 to 9, preferably 7.5. After this Homogenizing the mixture with a homogenizer will make the insoluble ones Components centrifuged. From the solution thus obtained, Zu administration of trichloroacetic acid to a final concentration of 2.5% active protein precipitated and removed by centrifugation. From that obtained solution, ether-soluble constituents are extracted by extraction removed and then the protein precipitated with cold acetone. The new Protein can be determined by ion exchange and pH gradient chromatography isolate from the precipitated product.  

The protein described here is concentrated in the ticks between 1-100 µg / kg. To use the protein for pharmaceutical purposes in To make larger quantities available, one can use known genetic engineering Methods (see Maniatis, T. et al: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1982). For this purpose first identified the genetic information for the new protein and the corresponding nucleic acid can be isolated. This becomes pure Protein reduced with dithiothreitol, then iodoacetamide becomes a derivative sation of the free SH groups added and then this is so traded protein with cyanogen bromide and trypsin split into small peptides. The The peptides are separated using reversed phase chromatography. The N-terminal sequencing of one of these purified peptides revealed that Sequence SDYEFPPPKKSRPG.

The existing peptide sequences now allow the synthesis suitable oligonucleotides a unique identification of the gene from the Genome or from corresponding c-DNA banks by sequence-specific Filter hybridization.

The genetic information thus obtained for the protein can then be found in various host cells, such as eukaryotic cells, yeasts, Bacillus Subtilis or E. coli expressed according to known methods and the protein can be obtained in this way. Arises in the eukaryotic cells the protein in glycosylated form.

The muteins, which are replaced by exchange, deletion or addition of amino Deriving acids or peptides from the new proteins are preferred represented by genetic engineering methods.

The new protein has anticoagulant properties and can be used Prevention and treatment of vascular diseases such as heart attacks, lungs embolism, arterial embolism and phlebothrombosis can be used. Continue are suitable for the preservation of blood. Beneficial for these uses is the low toxicity of the new protein.

The new protein is also suitable for immunizing grazing cattle against Tick-specific proteins. For example, the neutralizing antibodies can Food intake of the ticks may be disturbed. This turns the ticks into Leaving the landlord and thus dying.

Examples 1 to 3 Example 1 Purification of the thrombin inhibitor

A culture kept in the laboratory at 28 ° C and 80% relative humidity Leather ticks (Ornithodorus moubata) became in it every 14 days fed that they could suck on rabbits. Ticks of all development stages before or after feeding were frozen at -20 ° C and at the subsequent workup. 10 g ticks were used in 20 ml of phosphate-buffered saline (20 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.5 and homogenized at 20 ° C. The insoluble Ingredients were separated by centrifugation.

The clear, red supernatant was trichloroacetic acid (TCA) up to a Final concentration of 2.5% TCA added. A massive, red precipitate which was separated by centrifugation. After that was the solution was shaken 3 times with 1/10 of its volume of ether. The aqueous solution was neutralized with NaOH.

Four times the volume of cold (-78 ° C.) acetone was added. The solution stood on dry ice in the cold. It was formed in the course of a flaky, white precipitate for up to 24 hours. This was centrifuged off and dried in the desiccator. The yield was 1% of the weight of the used ticks. This product showed in SDS gel electrophoresis 10 to 20 protein bands in the low molecular weight range up to 40 kDa Coloring with Coomassie brilliant blue. The isoelectric point of this Proteins ranged between pH 4 and pH 7.

This protein mixture (200 mg) now became a protein concentration of 20 mg / ml in phosphate-buffered saline, pH 5, and added to a Q-Sepharose® column (Pharmacia Fine Chemicals, fast-flow) applied. The Volume of the column was 10 ml. The column was filled with the same buffer equilibrated in which the sample was also taken. After washing the Column with three column volumes of equilibration buffer was used with the column eluted with a pH gradient. For this, 10 column volumes of the wash buffers (pH 5.0) over a gradient mixer slowly with 10 columns volumes of wash buffer (pH 1 to 3) mixed. Under these conditions, elu the thrombin inhibitor at pH 4.5. The protein was so pure and get active form. It showed a molecular in gel electrophoresis weight of 15 000 ± 1000. The amino terminal sequence of this protein reads: SDYEFPPPKKSRPG. Its isoelectric point is at pH 4 to 5.  

Example 2 Separation of active and inactive thrombin inhibitor molecules

The homogeneous by molecular weight, obtained according to Example 1 and by Protein identified as thrombin inhibitor activity was as in Example 1 described by the 4-fold excess of cold acetone precipitated and ge dries. The residue was then placed in phosphate buffered saline, pH 7.5, added (protein concentration 20 mg / ml) and to a with the the same buffer of equilibrated Mono-Q column (column volume 1 ml) (about 0.1 mg). The column was at a flow rate of Washed 0.5 ml / min and then with a gradient of 150 mM NaCl 350 mM NaCl (2.5 ml each) and then from 350 mM NaCl to 450 mM NaCl (each 2.5 ml), followed by 450 mM NaCl after 550 mM NaCl (10 ml each) and close Lich eluted to 800 mM NaCl. The protein appeared between 350 mM NaCl and 550 mM NaCl. However, activity (measured as a thrombin inhibitor effect) only associated with an absorption maximum at 450 mM NaCl. In however, the sequence analysis of all eluted proteins showed that all have the same amino terminal sequence, thus with regard to their Primary structure are identical.

Example 3 Renaturation of the inactive thrombin inhibitor molecules

The inactive thrombin inhibitor fraction obtained in FIG. 2 was processed as in Bei game 1 is described and recorded in a renaturation buffer, which contained 8 M urea and 200 mM dithiothreitol (DTT). After 1 h at The protein was buffered at 37 ° C. against 100 times the volume of phosphate Saline dialyzed (exclusion volume of the dialysis tube 10 kDa). After 2 hours Dialysis at 4 ° C the protein was active and showed on the Mono-Q column Elution through a salt gradient has the same behavior as the active one Fraction in example 2.

Claims (3)

1. Protein, characterized by the N-terminal amino acid sequence SDYEFPPPKKXRPG, in which XS or N, and the molecular weight of about 15,000 Daltons, and its muteins. 2. Protein according to claim 1 for use in combating Diseases. 3. Use of the protein according to claim 1 for the production of Antibodies against tick infestation or to immunize grazing cattle against tick infestation.