生物专业英语综述作业Word格式.docx
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生物专业英语综述作业Word格式.docx
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ThesiteandthemechanismofoxidationofArgininekinase
Abstract
Argininekinase(argininekinase,AK)(EC2.7.3.3)isaphosphodiesteraseguanidineskinase,composedof403aminoacids,whichcontainsfivecysteines(Cys23,Cys127,Cys139,Cys201,Cys271),theexperimentwefoundthattheoxidationstatebandandrestorethestatewiththephenomenonofelectrophoresisAK,andthroughaseriesofbiotechnologymeans,respectively,ofthesefivesitesCysmutatedtoserine(Ser),toidentifyoxidationsitesandtostudytheoxidationmechanism,andbyUVfluorescence,respectively,toexplorethestructureafterthemutationofthetheAKvarioussitesrelatedtoactivity.
Keywords:
Argininekinase,rite-directedmutation,oxidationsites,SDS-PAGE
Introduction
1.Argininekinase[1](ArgininekinaseAK)(EC2.7.3.3)isakinaseofaphosphorylatedoriginalguanidinocompoundexistswidelyininvertebrates,andisanimportantkinasethathasadirectrelationshipwithintracellularenergyoperation,musclecontraction,ATP-regenerating.ItsFunctionishowtoCatalyticreversiblereactionsbythefollowingstep:
ATPphosphategroupistransferredtothearginine,therebyformingahighenergybondenergystoragemolecules-argininephosphate.
Thereactionequationisasfollows:
Mg2+ATP+ArgMg2+ADP+Argininephosphate
earlyin1998,MrZhouwithsomepeoplehaveusedthecrystalstructureofthetransitionstateAKfromhorseshoecrabsbyhorseshoetocrabtheLimuluspolyphemussinglesubunitAKdissociationcombinedtransitionstateanalogs.TheresultsshowedthattheAKwasmadeupbyasmallα-helixoftheN-terminaldomainandalargeC-terminalstructure(112,-357residues).TheC-terminaldomainisSimilartotheC-terminaldomainofglutamatesynthase,thetheBaguantiparallelβ-sheetisaroundbysevenα-helicalpackage[2].Seenfromthisstructure,thetransitionstateanalogisnotincorporatedinthemiddleofthetwodomains,butcombinedintheC-terminal'
sInteriorregion,whichmeansthattheAKistheactivesiteintheCterminal.Inaddition,theC-terminaldomaincanbedividedintotwosmallstructure,whileatransitionstateanalogsisincorporatedatthejunctionofthetwodomains[3].AccordingtoMr.ZouChenglu'
spointofviewthattheactivesiteoftheenzymeisinarelativelyflexibleportionandisthepartsofthestructure,andthisPositionismoresusceptiblefromtheimpactofadverseexternalfactorsthanotherparts,theresultisthattheenzymeactivitylossfasterandeasierthantheenzymeofthewholestructure.
2.Theoxidationofprotein
Differenttypesofoxidativedamageinproteinisanimportantfacterofthecauseofmanyseriousdiseases(suchas:
cancer,diabetes,neurodegenerativediseases)ThechaperoneproteinoxidativedamageofER,willleadtothemisfoldingandaggregationdiseasesinprotein,suchas:
AD,Parkinson'
sdisease(Parkinson'
sdisease,PD),amyotrophiclateralsclerosis(amyotrophicoflateralsclerosis,ALS),andHangextensionRatonchorea(Huntington'
sdisease,HD),andsenescence-relateddiseases,animportantcommonfeatureofthemistheabnormalproteininthebrainwithintheaccumulation.
IntheexperimentofelectrophoreticdetectionwithAKwefoundtheoxidationstatebandandreducedstateband.Revelatedbytheresearchmethodsofcreatinekinase(CK),wemake5sitesCysofAKmutatedtoserine(Ser),thenuseofaffinitychromatography,UV,fluorescence,suchasarangeofbiotechnologytoolsexplorethisstructureandrelatedactivityafterfiveCyspointmutation,respectively,lookingfortheoxidationofargininekinaselocusandtrytorevealthemechanismoftheoxidation,CyswasoxidizetoformtheS-Sdisulfidebonds,hydrophobicstructureexposed,sothatthered-shiftedfluorescencesweepconformation,sowemakemutationsinallfiveCyssites,hopingtopavethewayfortheproteinfoldingmechanism.
1Materialsandmethods
1.1Materials
1.1.1Materials
E.colim15(containingtherecombinantplasmidscarryingtheAKgenepQE60vector)
pET28bplasmid,E.coliDH5α(E.coliDH5α)
ExpressionstrainRosetta(providedbytherealSavelaboratory)
1.1.2Mainequipment
YC-1typethechromatographyexperimentsfreezer(BeijingBrainLABKangtheexperimentalapparatusDivision)
CXG-1-typecomputerthermostatchromatographycabinet(ShanghaitheHuxiAnalysisInstrumentFactoryCo.,Ltd.)
HD-3UVdetector(ShanghaiHuxiAnalyticalInstrumentFactoryCo.,Ltd.)
TGL-16LAtheGL-2Mtypefreezercentrifuge(HunanStarScientificInstrumentCo.,Ltd.)
GL-2Mtyperefrigeratedcentrifuge(HunantheStarScientificInstrumentCo.,Ltd.)
SnowMountainicemachine(ChangyangdianScientificInstrumentsCorporation)
UltrapurewatermachineAYJ1-0501-U(theYeeYoungEnterpriseDevelopmentCo.,Ltd.)
DH-2120lowtemperaturethermostatbath(theHaijiaPengTechnologyCo.,Ltd.)
ElectrophoresisinstrumentDYY-5(Beijingsixty-oneInstrumentFactory)
BS-1Eshakingincubator(JintanCity,JiangsuProvince,YitongElectronicInstrumentFactory)
SW-CJ-1FDSingleSidedCleanBench(SuzhouPurificationEquipmentCo.,Ltd.)
3FG-01BelectricthermostatBlastOven
AutoclaveYXQ-LS-50S(ofHaiboMotionLimited)
PCRmachine(Biometra,Germany)
PHmeter(ORION3STAR,Thermoorion)
Electronicbalance(TE412-L,CP64,GermanySatorinsCompany)
UVspectrophotometer(U-4300,Sweden/GE)
Fluorescencespectrophotometer(F-4500,Hitachi)
ProteinpurificationAKTA-purifieranalyzer(Sweden/GE)
Ultrasoniccellpulverizer(XO-650,NanjingfirstEuropeanBiologicalTechnologyCo.,Ltd.)
1.1.3Reagentsandsolution
KOD-Plus-MutagenesisKitToyoboBiology(Shanghai)TechnologyCo,Ltd;
synthesisandDNAsequencingofPCRprimerstocompletebytheShanghaiBiologicalEngineeringCo,Ltd.
Reagents:
Isopropylthio-β-D-galactoside(IPTG)
Sodiumdodecylsulfate(SDS)
Mercaptoethanol(BibtehnologyGrad)
His-nickelaffinitychromatographyresin(GEHealthcare)
AllotherreagentswereofanalyticalgradeMolecularbiologyinavarietyofcommonlyusedbufferedsolutionisformulatedsee"
MolecularCloning(ThirdEdition)Othermajorsolutionformula:
Weighing2.4228gTris-baseand29.25gNaCl,adddistilledwatervolumeto1L1L5*Bindingbufferbuffer:
HCLadjustedtopH8.0withdiluted1*bindingbuffer.MeasuringenzymeactivitySubstrate:
57mmArgat2ml,66mMMg(AC)2.0ml,thymolblueindicator2.0mlATP0.0532g,distilledwater14mlAddNaOHtopH8.2.Cystine.
1.2Methods
1.2.1FKBP12geneamplification
AKC23SUP
CCAAGTCTCTCCTTAAGAAGTATCTTAC
AKC23SDOWN
AGTCGGTGGCGGCTTCAAGTTTCTTGAAA
AKC127SUP
CCGGTCGCTCAATGCAAGGTTACCCCTT
AKC127SDOWN
AGCGGACTCGGGTGGAGATAACGAACTT
AKC139SUP
CTCACTGAGTCTCAGTACAAAGAGATGGA
AKC139SDOWN
GGAGGGGTTGAAGGGGTAACCCTGC
AKC201SUP
CCCGCTACTGGCCCGCCGGACGTGGAAT
AKC201SDOWN
AAGCGTTGGCTGCTTGCAGGAAGCGGT
AKC271SUP
AGTCCCACCAACCTTGGCACCACTGTGC
AKRC271SDOWN
GAAAGTGAGGAAGCCCAGGCGGTCGTG
1.2.2AK-wtandAK-mutantproteininducedexpressionandpurification
1.2.2.1activationofrecombinantstrain
RecombinantstraintheRosetta/AK-MutantandRosetta/AK-wtwereinoculatedinto6mlofLBmedium(amediuminclusivefinalconcentrationof50μg/mlKana)andculturedfor12hoursin37°
Covernight,shakingspeedof200rpm.
1.2.2.2expandingcultureandexpression
ActivationofthestrainstheRosetta/AK-themutantandRosetta/AK-wtvaccinationinKanacontaining300mLLBculturemediumconicalflask(mediumcontainafinalconcentrationof50μg/mL),37°
Cshakingculture2to3hourstoA600ofabout0.6to0.8,theRosetta/AK-MutantaddedIPTGtoafinalconcentrationof0.2mMplaced20℃shaker,180rpm,inducibleexpression,tocontinuetheculturefor12hours.TheRosetta/AK-wtaddedIPTGtoafinalconcentrationof0.5mMat37℃shaker180rpminducedexpression,theculturewascontinuedfor5hours.
1.2.2.3proteincrudeextract
(1)Thecellculturewascentrifuged,6000rpm,centrifugedfor10min;
(2)Thesupernatantwasdiscarded,thecentrifugalwallofthebacterialcellswasaddedacertainamount(about25mL)washbuffer;
(3)againbycentrifugation,6000rpm,centrifugationfor10min;
(4)Thesupernatantwasdiscarded,thebuffersolutioninacertainamount(about25mL)isaddedtothebacterialcells,resuspendedbacterialcells;
(5)diligentratio
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