Preparation Of Multifunctional ?-hemolysin Nanopore Single Molecules Sensors

Biological nanopore sensor can achieve the detection of single molecules on a microscopic scale,and has significant advantages at the single-molecule analysis,single-molecule chemistry reaction research and other fields.a-hemolysin(aHL),a water-soluble protein monomers secreted by staphylococcus aureus,can be assembled into a stable heptamer protein nanopore on the double lipid membrane and is an ideal nanoscale sensing device.Natural a-hemolysin nanopore is a non-selective open channel with stable structure,but the narrowest part of this nanotube is only 1.4 nm.Pore size and non-selective channels greatly limit its detection range and sensitivity.The number of positive and negative charge proportion in heptamer nanopores cavity,aperture size and structural characteristics can be changed by genetic engineering of a-hemolysin mutation and directional chemical modification.Cysteine is widely used as a directional mutation of amino acids due to the existence of thiol.It can be connected with specific ligands to change the pore space structure or function,expand the application of a-hemolysin nanopore in the single molecule detection.In this paper,multiple different amino acids in a-hemolysin were mutated to cysteine residues to study the impact of these sites for protein activity and polymerization.Secondly,we study the assembling of a-hemolysin protein on the monolayer membrane through DPhPC.The assembly of a-hemolysin monomeric protein has been conducted on bilayer membrane,and the mechanism researches are still unsatisfactory.This method provides a new way for monomeric protein assembly.The completion of this project will greatly expand the application of a-hemolysin nano-channels,and provide a useful exploration about controlled assembly and integrated function of membrane proteins,also provide the strong technical support for the wide use of nanopore detect in various fields.This paper includes the two parts as follow:1.The Preparation of Cysteine mutant a-hemolysin nanopore.The experiment explores the preparation of a-hemolysin and using site-directed mutagenesis to build cysteine mutant proteins of aHL as nanopore sensors.We undergo single cysteine mutation of wild-type gene of aHL at bits 17,18 of Top position,103,105 bits of Vestibule,113 bit of Constriction,117,121 bits of P-barre,and 128,129 bits of Bottom.By expression in bacteria and purified by ultrafiltration retentate tube,the monomeric protein was prepared and purified,then the activities and assemblyed capacities of different mutant were compared.The results showed that 103 and 105 bits of aHL limit the formation of nanopore.2.The study of assembly of a-hemolysin monomeric protein on monolayer membrane.In this study,heptamer nanopore was prepared by DPhPC monolayers,and the conditions of monolayer membrane assembly were explored.The thermal stability and punching ability of the heptamer(aHL7)D and(aHL7)R formed by a-hemolysin on the double layer film and monolayer membrane was compared.The results showed that both proteins are relatively stable under 65 °C.When the temperature rises,heptamer begins to decompose to monomeric proteins,and both proteins have no significant differences in the single-channel recording,showing that the method of using single-layer film to assembly nanopores is feasible.