Preparation method and application of ntd domain in conotoxin αd-gexxa and its optimized sequence
A conotoxin and sequence technology, applied in the field of biochemistry, can solve problems such as cardiac side effects and addiction, and achieve the effect of good application prospects
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Embodiment 1
[0076] Embodiment 1: the NTD preparation of conotoxin αD-GeXXA (chemical synthesis method)
[0077] The inventors found in the research that the conotoxin αD-GeXXA NTD (αD-GeXXA-NTD) also has inhibitory effect on acetylcholine receptors, for example, it has relatively strong inhibitory effect on α9α10 acetylcholine receptor subtype. Therefore, it is necessary to chemically synthesize the N-terminus of conotoxin αD-GeXXA mature peptide.
[0078] figure 1 It is a circuit diagram for the preparation of conotoxin αD-GeXXA-NTD in Example 1 of the present invention. Such as figure 1 As shown, in order to obtain the antiparallel NTD dimer, first chemically synthesize two polypeptides, the two polypeptide sequences are the amino acid sequence of 1-20 in αD-GeXXA. The sixth cysteine sulfhydryl group (-SH) of the first polypeptide (N6) is not protected, while the 18th cysteine sulfhydryl group is protected by Acm (acetamidomethyl, acetamidomethyl) (-S-Acm) . The sixth cysteine ...
Embodiment 2
[0085] Embodiment 2, the specific preparation steps of sNTD are as follows:
[0086] (1) The sulfhydryl group of the 6th cysteine of sN6 is activated by DTNB: 1 μg / μl of sN6 and 1 mM DTNB reacted in 150 mMPBS, pH 7.3, at room temperature for 15 minutes in the dark and then terminated with 0.1% TFA, and the reaction product was subjected to HPLC Purify and identify the molecular weight by mass spectrometry, and obtain the correctly folded product (the 6th cysteine of sN6 is activated by DTNB to sN6*); sN6 is figure 1 Remove the first and last ends (DVHRP and SL) of N6, which is CQSVRPGRVWGKC.
[0087] (2) The sixth cysteine of sN6 forms the first disulfide bond with the 18th cysteine of sN18: 0.4μg / μlsN6* and 0.3μg / μl sN18, in 150mM PBS, pH7.3, After reacting in the dark at room temperature for 15 minutes, stop it with 0.1% TFA, and the reaction product is purified by HPLC and identified by mass spectrometry to obtain a correctly folded product (sN6-sN18);
[0088] (3...
Embodiment 3
[0090] Example 3, the specific preparation steps of cNTD are as follows:
[0091] Synthesize a linear peptide of 27 amino acids. The crude peptide is first purified by HPLC. The purified peptide is dissolved in 50mM Tris HCl, pH 8.1, and the peptide concentration is 0.075μg / μl. It is oxidized in air at 4°C for 24h. After the reaction is completed, add 0.1 The reaction product was purified by HPLC and identified by mass spectrometry to obtain a correctly folded product (cNTD).
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