Polypeptides with function of self-assembled potassium channel and its application
A potassium channel and self-assembly technology, applied in the field of polypeptides with self-assembled potassium channel function, can solve the problem of lack of synthetic ion channel drugs, etc., and achieve the effects of high purity, small molecular weight and low cost
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[0024]The preparation method of the polypeptide of the present invention adopts a chemical synthesis method, that is, a very mature solid-phase peptide synthesis method well known in the art, and its amino protection strategies mainly include 9-fluorenylmethoxycarbonyl (Fmoc) method and tert-butoxycarbonyl (Boc ) method two, at present domestic and foreign mainly adopt Fmoc solid-phase synthesis method. The solid-phase synthesis method can use commercially available protected amino acids as raw materials to obtain a large number of samples quickly and efficiently. In the 9-fluorenylmethoxycarbonyl (Fmoc) synthesis system, preferably Wang (Wang) resin, add 15ml / g of dimethylformamide (DMF) for 30 minutes, then add a total of 20% of hexahydropyridine to treat at room temperature 20 minutes to remove the Fmoc protecting group, and then add an excess of the corresponding protected amino acid and the activator benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), ...
Embodiment 1
[0037] A polypeptide with self-assembling potassium channel function is a polypeptide containing 24 amino acids: the primary structure of the complete sequence of the polypeptide is: leucine-tyrosine-leucine-alanine-valine-formazine Thionine-Valine-Phenylalanine-Alanine-Leucine-Valine-Leucine-Glycine-Tryptophan-Methionine-Asparagine-Alanine-Leucine Acid-tyrosine-phenylalanine-threonine-arginine-glycine-leucine (the amino acid sequence of the polypeptide is: Leu-Tyr-Leu-Ala-Val-Met-Val-Phe-Ala -Leu-Val-Leu-Gly-Trp-Met-Asn-Ala-Leu-Tyr-Phe-Thr-Arg-Gly-Leu).
[0038] Polypeptide of the present invention (Leu-Tyr-Leu-Ala-Val-Met-Val-Phe-Ala-Leu-Val-Leu-Gly-Trp-Met-Asn-Ala-Leu-Tyr-Phe-Thr-Arg-Gly- Leu) solid-phase synthesis
[0039] The polypeptide of the present invention whose sequence is LYLAVMVFALVLGWMNALYFTRGL was synthesized by a commercially available Quartet polypeptide synthesizer (PTI company), and the specific steps were as follows:
[0040] 1. Add appropriate amount...
Embodiment 2
[0047] Polypeptide of the present invention (Leu-Tyr-Leu-Ala-Val-Met-Val-Phe-Ala-Leu-Val-Leu-Gly-Trp-Met-Asn-Ala-Leu-Tyr-Phe-Thr-Arg-Gly- Leu) forms K on lipid bilayer membranes + ion single channel current
[0048] 1. Materials and methods
[0049] 1.1 Materials: KCl and NMDG-Cl were purchased from Sigma Company, POPC phospholipids and phosphatidylserine (phosphatidylserine, PS) were purchased from Avanti Company (Avanti Polar Lipids, USA), inverted microscope (IX70, Olympus, Japan), and microelectrodes were drawn instrument (P-97, Sutter, USA), three-dimensional microelectrode manipulator (MP-285, Sutter, USA), patch clamp amplifier (EPC-9, HEKA, Germany), PULSE+PULSEFIT recording and analysis software (HEKA, Germany).
[0050] 1.2 Synthesis of lipid bilayers: Weigh 80 mg POPC and 20 mg phosphatidylserine, dissolve them in 2 ml double distilled water, suspend with a vortex mixer for 20 minutes under nitrogen, and then ultrasonically emulsify for 10 minutes. Centrif...
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