| ATP synthase,or FoF1-ATPase is a key enzyme in cellular energy metabolize.It is a complex with several subunits and widely distributed in almost all kinds of organisms. According to its polarity,it can be divided into two major sections which are hydrophilic section outside the membrane and hydrophobic section on the membrane, respectively.This characteristic makes its purification become complicated as well.In this thesis,we managed to purify the enzyme in two different ways:in the first method,a plasmid which contain the complete sequence of ATP synthase complex is constructed,in which a His tag is added into the subunit c by the mutation.Then, purify the tagged protein by Ni2+-NTA affinity chromatography.The second is to design a special method for a wild type thermophilic called Thermomicrobium roseum.After the selection and cultivation at a high temperature,we crush the cell membrane by ultrasonic and collect the chromotaphores as the intermediate by centrifugation.The chromotaphores can be used either to purify the protein or the IBR construction.According to its structure and catalytic mechanism,ATP synthase can also be recognized as a combination of two sections-the stator(ab2δα3β3) and the rotor(γεcn) -functioning together.Research showed that different load combined to theβsubunit can cause different result during the ATP synthesize.When getting the pure protein with biological activity,we constructed an immuno-rotary biosensor(IRB) based on FoF1-ATPase with immunological and biochemical techniques.The construction, TF1βantibody-biotin-streptavidin-biotin-CL antibody can be used to binding the specific antigen.By detecting the signal of fluorescence probe which is affected by the H+ in the process of ATP synthesize,the detection become more sensitive.We also try to use the QDs with different wavelength to encode in a simple way which is a beneficial attempt in practical application and obtain some valuable results.
|
PDF Full Text Request