Screening And Expression Of Micromolecule Polypeptide

Acid-sensing ion channels?ASICs?are proton-gated cation channels that are widely expressed in peripheral and central nervous systems.ASIC1a is an important subunit member of the acid-sensitive ion channel family and is involved in many physiological and pathological processes in the body,such as synaptic transmission and development;learning and memory;fear and depression related behaviors and other physiological processes,pain,inflammation,seizures,multiple sclerosis and many other pathological processes involved in tissue acidosis.Based on the physiology and pathology of ASIC1a,ASIC1a inhibitors have very important applications in the development of effective analgesic drugs,neuroprotective drugs,or drug design leader molecules,so how to obtain or modify ASIC1a inhibition with high selectivity and high activity have been a hot topic in neurobiological research.In this study,a random peptide library was constructed in the framework of ASIC1a selective inhibitor PcTx1.At the same time,using ASIC1a as a target,the active polypeptides with ASIC1a regulation or inhibition were screened in this random polypeptide library by yeast two-hybrid method.So as to establish an efficient and convenient screening system for obtaining ASIC1a inhibitors.For the above purpose,this study mainly completed the following three parts of the work.First,a random pentapeptide library with a library capacity of 3.05×10⁶ was constructed in the framework of PcTx1 by PCR.In random library abundance tests,30clones were randomly selected for sequencing.The sequencing results showed that 90%of the sequences in the random library were in line with the original intention of the experimental design,and there were no repeat sequences in the 30 clones,which proved that the random library constructed had a good abundance.Second,using a yeast two-hybrid method to screen for a polypeptide molecule that may interact with the extracellular region of rASIC1a from a library of random pentapeptides constructed using PcTx1 as a framework,using the extracellular region of the rASIC1a isoform ion channel as a“bait”.Through a round of yeast two-hybrid screening,a total of 44 positive clones were obtained on a yeast-deficient medium plate?-Trp/-Leu/-His/-Ade medium plate?.Analyzing the amino acid sequences of these 44 positive results,five plasmids?L9;L23;L24;L25;L37?met our original design of the experiment,except that the mutations at the five key amino acid residue positions were different from those of PcTx1.And the amino acid residues basically retained the ICK model of PcTx1.The remaining 39 plasmids were frameshifted during randomization,resulting in a polypeptide consisting of 57-61 amino acid residues.Most of these peptides contain a conserved sequence of 27 amino acid residues.Thirdly,the selected plasmids?L9;L23;L24;L25;L37?were inserted into the expression vector pET43-His-SUMO using the MP cloning seamless cloning method to construct the corresponding expression plasmid.The fusion protein was induced to express in E.coli Shuffle^TM by auto-induction expression,and the fusion protein was purified by affinity chromatography using the Ni column.The purified fusion protein used SUMO protease 1 to cleave the SUMO tag and release the corresponding polypeptide molecule.After the polypeptide molecules are released,the polypeptide molecules are further purified by reversed-phase liquid chromatography?RP-HPLC?to obtain the high-purity polypeptide molecules which can be used for physiological activity detection.Identification by MALDI-TOF mass spectrometry revealed that the identified molecular weights of L9;L23;L24;L25;L37?whose molecular weights were 4520.7896 Da,4383.4268 Da,2965.5471 Da,4518.2383 Da,and 4520.6313 Da?were identical to their theoretical molecular weights.?The theoretical molecular weights thereof are 4520.19Da,4383.31 Da,2965.91 Da,4518.18 Da,and 4520.19 Da.?In addition,some of the polypeptide molecules were also tested for physiological activity,and it was determined that the polypeptide molecule L37 can inhibit 20%the channel current of rASIC1a at a concentration of 1?M.In summary,this study establishes an efficient and convenient screening system for obtaining ASIC1a inhibitors,and also provides an expression system suitable for polypeptide molecules containing multiple pairs of disulfide bonds.This greatly simplifies the process and steps for obtaining or modifying highly selective and highly active inhibitors of ASIC1a,thus provides some theoretical support and material basis for the development of ASIC1a target ligands and the development of related drugs or drug leaders.