Ntsiab lus:

Tsab xov xwm no rov xaiv ob yam tseem ceeb uas yuav tsum tau txiav txim siab hauv cov ntawv thov protein, cov kev xav tau ntawm cov protein ntawm cov protein lawv tus kheej. Cov ntawv qhia kom paub tias kev ua kom muaj protein zoo protein yog lub hauv paus rau kev ntseeg tau thiab rov ua dua ntawm cov roj ntsha proteins. Cov xwm txheej sib txawv muaj cov kev xav tau rau cov tshuaj lom neeg, cov yam ntxwv endotoxin, thiab cov muaj protein ntau tuaj yeem cuam tshuam rau cov tswv yim ua kom huv siab. Raws li cov xwm txheej tshwj xeeb, cov lus piv txwv tias cov proteins tshwj xeeb yuav tsum tau txais cov qauv ua kom huv si. Thaum kawg, cov txheej txheem protein ntau lawm tau npaj siab los ntawm kev xaiv cov qauv ntawm biophyity (xws li Sec-Mals, DSF, thiab lwm yam). Tsab xov xwm no ua haujlwm los muab cov lus qhia tseeb rau cov kws tshawb fawb, pab lawv ua cov protein, yog li ua tau zoo ntawm kev tshawb fawb zoo thiab ua tau zoo ntawm kev tshawb fawb.

 

Daim duab 1. Schematic Daim duab ntawm Cov Txheej Txheem Protein Ntau Lawm

 

Kev Xav Tau Cov Protein Siab - Piv Txwv ntawm Cov Teeb Meem Txog Cov Teeb Meem, Kev Siv Hluav Taws Xob thiab Kev Tawm Tawm

 

1.nucleic acid khi rau protein:

Thaum purifyic nucleic acid khi cov protein, ntau cov kauj ruam yuav tsum tau tshem nucleic acid paug. Thaum lub sij hawm lysis, nucleiges (xws li sm nucastase (Benzonase®) lossis DNZONASE®) lossis RNZONASE) yuav tsum tau ntxiv, tab sis ob lub nucleic acids tsis tuaj yeem raug tshem tawm kiag li. Hloov nucleic acid tshem tawm, xws li Pei lossis Streptomycin sulfate nag lossis daus, headinin purification lossis ion-txig chromatography. The presence of nucleic acids was monitored by the ratio of A260nm/A280nm throughout the purification process. Yog tias qhov piv tau qis dua 0.6, nws qhia tau tias cov protein purity yog tus muaj kua qaub heev thiab nucleic acid paug tau tsawg. Rau cov protein nrog muaj zog khi nucleic acids, lawv tuaj yeem raug txwv tsis pub ib ntus (xws li kho nrog urea) thiab tau renaturated. Cov ntsiab lus tseem ceeb: Siv cov tshuaj lom neeg tseem ceeb, cov buffs tshiab thiab cov kab huv (huv nrog 0.5m naoh ua ntej siv).

 

2. Niag Ferritin Hnyav saw:

Lub hom phiaj ntawm kev ua kom huv si nas ferritin hnyav saw (mfth1) protein yog high-particle cryo-electron tsom iav (Cryo-em). Tus Escherichia Coli qhia vector encoded unrabeled mfth1 yog ultrasonically tsis yooj yim yam tsis muaj kev ntxiv ib qho nuclease. Tom qab cua sov ntawm 7 0 degree, nws underwent ammonium sulfate daus, lim thiab tsis muaj kev cais tawm cheem muag. Qhov tshwm sim tshwm sim pom tias muaj ntau cov kev paug loj hauv Cryo-Electron Cov Lus Cim Ua kom zoo cov kauj ruam. Ntxiv SM Nuclease thaum lub cell cov txheej txheem thiab cov txheej txheem lim dej tom qab daus, thiab tom qab ntawd}} nm / A280NM los ntawm 1.4 txog 0.8. Tom qab loj me ntsis tsis pub tawm cov chromatography, qhov sib piv ntawm A260NM / A280nm ntawm MFH1 qauv kawg yog 0.56. Lub SDD-nplooj ntawv thiab Cryo-electron cov duab tsom iav tshiab (Daim duab 2B) pom tias cov protein tau pom tias cov protein yog qhov tseeb heev, cov paug tau raug tshem tawm.

Daim duab 2 Nas Ferritin Hnyav saw

 

 

 

3. Neeg muaj protein protein tib neeg dsrbec (dsrbd-egf- chimera):

DSRBEC yog tsim los ntawm kev fusion ntawm ob-stranded rna Thaum DSRBD tau qhia tawm hauv Escherichia Coli, nws qhia tau tias DSRNA siab heev. Tom qab cov lysis cell, cov kab mob cell, cov kab mob tsis zoo yuav cuam tshuam cov kev khi ntawm cov tshuaj tiv thaiv ntawm cov tshuaj tiv thaiv kev ruaj khov rau cov hlau tsau chromatography (IMAC) kem. Cov hau kev pab cuam xws li Pei lossis Streptomycin sulfate nag lossis daus, rnase kev kho mob lossis cov kev daws teeb meem siab tsis tuaj yeem tshem tawm RNA. Lysis ntawm lysis yog ua siv tsis muaj qhov tsis muaj 4m urea. Tus supernatant tau thauj khoom rau IMAC kem, thiab 4m rau {{}} m urea tau maj mam siv thaum hmo ntuj (kem renaturation). Lub renaturation ntas tau ntxiv nrog imidazole thiab eluted los ntawm IMAC kem. Lub chaw ua kom zoo kawg yog nqa los ntawm SuperDex 75 gel Pom kev kom tau txais DSRBEC.

 

4. Cov proteins khi rau cov chaw sib txawv lossis lwm yam chofactors:

Rau cov protein uas cuam tshuam nrog cov chaw sib txawv xws li zn {}}, lom} {2}}, lossis lwm yam cofactors) thaum lub sijhawm qhia. Ib qho me me ntawm tib cov kev sibtawm (lossis lwm qhov chaw ua haujlwm) tseem yuav tsum tau thaum lub sijhawm ua haujlwm. Txawm li cas los xij, kev siv ntawm cov neeg sawv cev chelating (xws li Edta, piv txwv li) thiab chelating txo cov neeg sawv cev (xws li DTE) yuav tsum zam. Thaum cuam tshuam nrog cov protein khi rau cov chaw sib xyaw ua ke tsis muaj cov neeg muaj txiaj ntsig (xws li lwm yam kev ua haujlwm ntawm cov neeg muaj zog (xws li attract cov protein)

 

5. Recombore Ferridoxin (RFD) muaj ib qho [2fe ± 2s] Cyakobicia mastigocladus laminosus

Ferridoxin yog lub soluble hlau-timulfur protein uas koom nrog kev hloov khoom hauv tshuab hluav taws xob. Nroj hom Ferroredoxin nqa ib qho [2FE ± [2s] COLENTICSTHED Secombyem I. Tus EscchericS RFD protein hauv Daim Ntawv Nruab Nrab ntawm TBD hauv Daim Ntawv Qhia TBD) thiab tshuaj tua kab mob. Kev lees paub ntawm Imac Capture tau ua kom siv lub ntas tsis muaj 10 hli histazole thiab tiv thaiv kev puas tsuaj ntawm [2fe ± 2s] pawg. Kev sib pauv pauv thiab tshem tawm cov chromatography tau siv rau kev ua kom huv si thiab muaj 12mg / ml rau crystallization kev tshuaj xyuas. Piv nrog rau kev ua kom zoo dua ntawm ferroredoxin, lub ntuj ferroredoxin huv los ntawm xiav-ntsuab alge mastigocladus ua tiav kev daws teeb meem siab dua thaum lub sijhawm crystallization.

 

6. Cov protein siv raws li antigens

Cov protein feem ntau siv los ua kom antigens los rov qab cov hom phiaj-spigands. Thawj qhov uas yuav tsum tau xav txog yog tias cov protein yog siv rau Vivo kev tiv thaiv lossis Polyclonal IGG, lossis rau cov camel icel lossis hauv cov txheej txheem vitro.

 

6.1 antigens siv rau kev tsim tawm cov tshuaj tiv thaiv

Thaum antigens yog siv los tsim monoclonal iGG antigen ntawm cov qauv ntawm cov kab mob uas tsis tau cuam tshuam nrog cov tshuaj protigens (xws li suav cov roj ntsha (xws li suav lub cev nqaij daim tawv) ua tiav. Txawm li cas los xij, txoj kev coj dawb huv yuav tsum raug tswj kom nruj kom tsis txhob muaj cov kab mob immunogen muaj zog cuam tshuam nrog lub cev tiv thaiv kab tiv thaiv kab xeb thiab ua rau cov hlau tsis muaj hom phiaj.

 

6.2 Kev soj ntsuam ntawm lub tsev qiv ntawv Conjugate

Saib xyuas tshwj xeeb yuav tsum tau them rau kev tswj hwm lub ntuj kev sib txuam ntawm antigen thaum lub sijhawm ua kev txhaj tshuaj NanoDyBodization. Tsis zoo li cov tshuaj tiv thaiv kab mob, cov tshuaj pleev xim Camel (tshwj xeeb yog Nanobodies) zoo li paub txog lawv cov cim convex complementary chaw. Ib yam li ntawd, thaum kuaj xyuas cov hluavtaws loj lossis cov antibody cov tsev qiv ntawv hauv vitro, cov antigen yuav tsum tswj tus qauv uas tau ua tiav nrog daim ntawv thov phiaj. Txij li thaum tus txheej txheem tshuaj ntsuam nws tus kheej yog tsis-kev sib thab, yog tias tus antigen tau ua rau cov neeg tsis sib haum xeeb

 

 

 

7. Cov protein muaj cov interbolecular lossis intramolecular disulfide lossis dawb cysteine

Cov ntawv sib thooj yog qhov tseem ceeb heev rau cov stabilizing lub ntuj qauv ntawm cov protein. Thaum lub sij hawm cov txheej txheem lim dej, thaum ua kom huv cov protein uas muaj cov neeg tsis sib haum xeeb los tiv thaiv cov protein ntau, yog li hloov lawv cov haujlwm. Rau cov protein muaj cov neeg sawv cev dawb, txo cov neeg sawv cev yog qhov tseem ceeb nyob rau hauv tag nrho cov kauj ruam ntawm cov txheej txheem lim dej, tshwj xeeb tshaj yog thaum muaj kev tsim ntawm cov khoom lag luam tsis zoo. Cov neeg sawv cev siv ntau tshaj plaws yog dithiothreitol (DTT), -mer), thiab tris (2- retboxyethyl) phosphine hydeblokyethethide (trep). Rau iMac resins uas tsis sib xws nrog DPT lossis TCEP, nws raug nquahu kom siv -me thiab hloov lawv nrog lwm cov neeg sawv cev rau cov kauj ruam ntxiv.

 

8. Cov tshuaj tiv thaiv cov tshuaj tiv thaiv kab mob

Txawm hais tias tus qauv ntawm cov tshuaj tiv thaiv cov tshuaj tiv thaiv kev siv tshuaj tiv thaiv (xws li nanobodies) yog yooj yim dua li ntawm Idg, lawv txoj haujlwm yog nyob ntawm qhov tseeb tsim ntawm kev cuam tshuam ntawm kev puas tsuaj. Nyob rau hauv cov kab ke hais tawm cov kab nruab, txawm nrog kev saws ntawm cov txheej txheem txhim kho, misfolded lossis ib nrab cov khoom ntim tau tshwm sim, uas muaj nyob rau hauv lub soluble thiab nyob rau hauv lub cev cais. Tshaj tawm yog tias cov tshuaj tiv thaiv kab mob kom nruj yuav ua rau soluble aggregates (colloid aggregation) los ntawm hydrophobic plaques nyob rau saum npoo. Xws li cov kev sib sau ua ke yog qhov nyuaj rau kev txheeb xyuas hauv kev ntsuam xyuas niaj hnub ua haujlwm (tab sis lawv lub peev xwm cuam tshuam tsis zoo rau cov ntawv sau tsis zoo uas tsis muaj kev cuam tshuam me me. Yog li ntawd, cia siab ib leeg ntawm SDS-nplooj yog tsis txaus los ntsuas cov qauv zoo. Cov hau kev biophysical xws li gel pom (daim duab 3) yuav tsum tau saws kom paub qhov txawv monomers (cov ntsiab lus tseem ceeb) los ntawm kev sib sau ua ke). Cov ntsiab lus tswj tau suav nrog: Optimizing Redox ib puag ncig lub sijhawm ua kom muaj kev cuam tshuam kev sib raug zoo, thiab txhim kho kev cia siab tom qab kev sib sau ua ke. Cov kev ntsuas no yog qhov tseem ceeb heev rau kom ntseeg tau cov monodispersity thiab muaj nuj nqi ntawm cov tshuaj tiv thaiv kab hlau tawg.

Daim duab 3. Gel Kev Pom Kev Sib cais ntawm Soluble Aggregates ntawm Nanobodies

 

9. Soluble tawg ntawm lymphocyte receptor llt1

Rov qab siv cov protein uas tsis muaj txiaj ntsig ntawm cov neeg muaj ntshauv (xws li lymphocyte receptor llt1) feem ntau ntsib cov teeb meem folding teeb meem. Gel pom ntawm qus-hom Llt1 qhia kev sib sau ua ke (Daim duab sab nrauv), tab sis cov duab loj tshaj plaws tshwm sim los ntawm cov cysteine ​​tsis tuaj yeem ua rau lub dimer (Daim Duab 4B). Rau lub hom phiaj no, ob tus merants tau tsim: ib qho kev tawm ntawm cov teeb meem cysteine, uas ua rau kev poob sai sai hauv kev tawm los (Daim duab 4A); Tom qab cov lus qhia ntawm neocysteine ​​kom rov tsim kho cov ntawv foob tsis zoo, tsis muaj qhov sib txuam zoo, tab sis kuj tseem tuaj yeem tsim cov ntawv raug cai thiab tswj tau lub ntsej muag zoo nkauj. Qhov xwm txheej no tau qhia txog kev tsim qauv kev tsis sib haum xeeb, ua ke nrog cov roj ntsha tsis sib xws, thiab cov tshuaj tiv thaiv kev ua haujlwm zoo nrog cov qauv sib luag thiab cov qoob loo ruaj khov tuaj yeem tau txais.

Daim duab 4. Disulfide Kev Tsim Kho Kev Tsim Kho Kom Yooj Yim Txhim Kho Cov Khaub Ncaws thiab Tawm Los ntawm Soluble Llt1

 

 

 

10. Yooj yim compertegable protein

Lub purification ntawm reebic proteins feem ntau ntsib cov teeb meem ntawm kev sib cav sib ceg, thiab tom qab ntawd ua raws li qhov "nucleation solub sib nraus ua ntej thiab tom qab ntawd ua raws li cov khoom sib cav sib ceg Txhawm rau daws qhov kev sib tw no, ntau hom kev xav tau los ntawm kev ua haujlwm siab, thiab hloov cov lus qhia txog kev ua kom zoo, thiab hloov kho cov kab mob ntau dhau, thiab hloov kho cov kab mob txawv txav ntawm IMAC rau SEC) yuav tsum tau saws kom sai sai tshem tawm cov nuclei. Feem ntau hais lus, thaum kev soj ntsuam cov kev daws teeb meem tsis zoo, yam xws li muaj feem sib xyaw, PE nqi, cov khoom siv tsis sib xws yuav tsum tau txais kev txiav txim siab (Daim Duab 5). Los ntawm kev ua kom zoo ntawm orthogonal nrhiav tau (xws li SEC, CD, DSF thiab qhov kub thiab lwm yam), cov protein zoo thiab cov kev daws teeb meem zoo tshaj plaws tau txiav txim siab.

Daim duab 5. Cov tswv yim rau cov protein tag nrho

 

11. Crystallization ntawm Tib Neeg Clk1 Kinase (Yuav ua li cas thiaj li tau txais cov khoom ua kom zoo nkauj)

Txhawm rau kom tau txais homogeneous tsis-phosphire vollated clk1 Kinase rau crystallization kev kawm, ntau lub tswv yim sib koom ua ke. Firstly, nws tau sib koom nrog λ-aws nrog λ -Phospatase hauv Escherichia Coli los tshem tawm cov heterogeneity ntawm autophosphorherlation. Txawm hais tias qhov txiaj ntsig tau raug txo kom tsawg, ua tiav dephosphaieldation tau ua kom tiav. Tom qab ntes ntawm iCac, tus Eluent tau txhawb nrog cov txhab ruaj khov (50mm DTT Tom qab ntawd, qhov tseeb oligomer tau sib cais los ntawm sec (muaj 5% glycerol thiab -me). Qhov kawg kev sib pauv qib sib pauv qib cais ntawm crystalline (tsis-phosphorylated) thiab tsis yog-crystalline (ib nrab phosphorylated) clk1 pawg. Nws yog tsim nyog tshwj xeeb tshaj plaws tias hom ntawm cell liysis cuam tshuam rau cov muaj protein ruaj khov ntawm clk1, qhia qhov tseem ceeb ntawm kev kho mob me me rau Kinease npaj.

 

 

 

12. Cov khoom lag luam nrov nrov -1} nrog ruaj khov ligand lub peev xwm

Txhawm rau npaj ua haujlwm galectin -1}}}}}, tsis muaj npe thiab nws daim ntawv tsis muaj txiaj ntsig thiab tsis muaj nuj nqis thiab tsis muaj kev sib cav sib ceg. Cov lus pom tseem ceeb qhia tau hais tias lactose letification chromatogrification tuaj yeem ua rau kom raug cov protlysis (hepes Tsiaj qus-hom Galectin -1 yog nquag mus rau oxidation thiab inactivation, thiab nws cov kev ruaj khov tseem ceeb nyob rau hauv lub xub ntiag (Daim duab 6). Txawm li cas los xij, cov cysteine-dawb mutant tseem ceeb heev thaum tswj kev sib piv ntawm NanodSf, ITC, thiab lwm yam) los ntawm kev tshem tawm kev ua cim sib xws.

Daim duab 6. Lub hydrodynamic tus cwj pwm ntawm recombant Gallein -1}} qhia nws qhov tsis muaj peev xwm

 

13. Endotoxin tshem tawm

Txoj kev tshem tawm tshem tawm endotox yog nyob ntawm affinity chromatography suav nrog kev sib txuas lus chratograph (pll), thiab ntxiv rau surfactant triton x {2}}. Thaum lub sij hawm cov txheej txheem lim dej, them sai sai rau kev npaj cov dej ntas tsis pub muaj lps Rau endotoxin paug, cov kua chromatographic twj / cov kua dej tuaj yeem tsim kom muaj kev vam meej nyob rau hauv {}}. {} M na a. Tus nqi kawg ntawm LPS hauv cov qauv tau ntsuas siv Lowulus Amebocyte lysate Reegent (lal), thiab nws tau qhia meej tias nws yuav tsum muaj rau daim ntawv thov tshwj xeeb.

 

14. Protein complex

Cov lus qhia thiab purification ntawm protein complex yuav tsum tau ua kom zoo raws li cov xwm txheej tshwj xeeb. Cov kev sib tw suav nrog qhov tsis txaus ntseeg lossis misfolding ntawm cov subunits. Txhua tus subunit tuaj yeem hais tawm ntawm nws tus kheej thiab sib sau ua ke hauv vitro, lossis co-qhia los ua cov protein ua haujlwm. Cov kev tsim tsim tsim yuav tsum ua tib zoo qhia nrog cov ntawv sau ua kom huv lossis kuaj kom tsis txhob cuam tshuam nrog kev sib txuas lus nrog kev sib txuas lus, tshwj xeeb tshaj yog thaum cov lus qhia nyuaj yog qhov yuav tsum tau ua. Thaum lub sij hawm lub zog lim dej, kev ncaj ncees thiab kev ruaj khov ntawm cov complex yuav tsum tau ntsuas. Cov hau kev suav nrog SDS-nplooj ntawv (Secunit Nrhiav Kom Tau), Sec (Homogeneity), Kev Ntsuam Xyuas Nyiaj Txiag Nws yuav tsum raug sau tseg tias cov complex yuav nthuav tawm ntawm cov kev ua kom muaj ntau. Nyob rau lub sijhawm no, cov qauv chratography lossis tsis sib xws (xws li dhau los ntawm kev ntsuas hluav taws xob drift lossis DSF kev ntsuas) yuav tsum tau ua kom zoo. Ntxiv mus, txo cov kauj ruam lim dej tuaj yeem tiv thaiv kom cov poob tsis muaj zog kev sib cuam tshuam thiab sib npaug ntawm kev ua kom muaj txiaj ntsig zoo thiab kev ncaj ncees ntawm cov complex.

 

 

 

15. Purification ntawm antigen-antibody complexes

Tshuaj tiv thaiv kab mob antibody-antigen complexes yog cov piv txwv ntawm cov protein complex. Lawv co-crystallization tuaj yeem qhia cov qauv khi thiab taw qhia qhov ua kom zoo ntawm antibody corpineering. Cov txheej txheem ib txwm muaj kev ua kom huv huv ntawm antigens thiab cov tshuaj tiv thaiv thiab tom qab ntawd sib tov lawv. Txawm li cas los xij, cov kev tshawb fawb tsis ntev los no tau qhia tias kev qhia sib txuas thiab cov laj kab ntsig txog cov kev ua kom huv thiab muaj txiaj ntsig zoo dua. Tsuas yog ib qho kev ua kom huv yog xav tau kom tau txais cov nyom, thiab tib lub sijhawm, tsis ruaj khov los ntawm cov tshuaj tiv thaiv, thiab txawm tias daim ntawv lo-dawb qhia tau ua tiav. Hauv qhov xwm txheej tshwj xeeb no, SDS-nplooj ntawv thiab gel pom (sec) feem ntau yog txaus los ntsuas cov kev coj dawb huv thiab qhov zoo ntawm cov complex.

 

Lub ntsiab

Kev ua kom muaj protein yog cov thev naus laus zis hauv kev tshawb fawb. Kev Kawm Txuj Ci Ntau Tshaj Plaws Feem Ntau Cov Qauv Txheej Txheem Millaigle, uas yog siv dav hauv cov txheej txheem biology, biochemistry thiab kev tshawb fawb ua haujlwm. Txawm li cas los xij, cov ntaub ntawv tshwj xeeb ntawm cov kev siv hauv qab dej (xws li qhov kev xav tau ntawm cov protein) lossis tsis muaj qhov cuam tshuam rau cov tsiaj uas yooj yim acid Qhov kev tshuaj xyuas no, hauv kev teb rau cov tswv yim tshwj xeeb no, cov qauv tsim kho kom zoo dua qub (xws li SEC, kev kuaj pom kev ua si, thiab lwm yam) ntawm cov kauj ruam tseem ceeb. Qee cov ntawv thov tseem yuav tsum tau xam cov kev txheeb xyuas ntxiv (xws li cov lus qhia acidox acid acid acid Los ntawm kev tsim cov qauv kev tswj hwm zoo thiab qhia meej txog cov tshuaj tiv thaiv protein ntau, thiab pheej sib kis ntawm kev tshawb fawb thiab cov qauv kev lag luam.

 

DOI: 10.1186 / s 12934-022-01778-5