蛋白表達(dá)及純化課件

1、蛋白表達(dá)、純化 抗體制備應(yīng)用及ELISA間接法盧士強(qiáng) 2010-11-18Part 1: 蛋白表達(dá)、純化蛋白表達(dá)流程目的基因cDNA表達(dá)宿主載體設(shè)計(jì)引物連接轉(zhuǎn)化 誘導(dǎo)表達(dá)蛋白純化鑒定保存geneCell-freeBacterialYeastInsectMammalianHost一：大腸桿菌表達(dá)外源基因的優(yōu)勢(shì)全基因組測(cè)序 , 共有4405個(gè)開(kāi)放型閱讀框架基因克隆表達(dá)系統(tǒng)成熟繁殖迅速、培養(yǎng)簡(jiǎn)單、操作方便、遺傳穩(wěn)定被美國(guó)FDA批準(zhǔn)為安全的基因工程受體生物大腸桿菌表達(dá)外源基因的劣勢(shì)缺乏對(duì)真核生物蛋白質(zhì)的復(fù)性功能缺乏對(duì)真核生物蛋白質(zhì)的修飾加工系統(tǒng)內(nèi)源性蛋白酶降解空間構(gòu)象不正確的異源蛋白細(xì)胞周質(zhì)內(nèi)含有種類(lèi)

2、繁多的內(nèi)毒素(endotoxin)Vector可使外源基因高水平表達(dá)的最佳啟動(dòng)子必須具備以下幾個(gè)條件1 ) 強(qiáng)啟動(dòng)子，外源基因的蛋白質(zhì)的表達(dá)量占細(xì)胞總蛋白的10% - 30%以上2 ) 應(yīng)能呈現(xiàn)出一種低限的基礎(chǔ)轉(zhuǎn)錄水平3 ) 應(yīng)該是誘導(dǎo)型的, 能通過(guò)簡(jiǎn)單的方式,使用廉價(jià)的誘導(dǎo)物得以誘導(dǎo)。常用的大腸桿菌表達(dá)載體啟動(dòng)子： 噬菌體的PL啟動(dòng)子 大腸桿菌乳糖操縱子lac啟動(dòng)子 色氨酸操縱子trp啟動(dòng)子 pBR322質(zhì)粒的beta內(nèi)酰胺酶啟動(dòng)子啟動(dòng)子終止子a: 如果在克隆基因編碼區(qū)的3末端之后，接上一個(gè)有效的轉(zhuǎn)錄終止子，便能夠阻止轉(zhuǎn)錄通讀過(guò)位于下游另一個(gè)啟動(dòng)子b: 如果在啟動(dòng)子的上游部位放置一個(gè)有效的轉(zhuǎn)

3、錄終止子 , 那么由該啟動(dòng)子驅(qū)動(dòng)的克隆基因的轉(zhuǎn)錄便會(huì)被限制在最低本底水平。c: 轉(zhuǎn)錄終止子還能增強(qiáng)mRNA分子的穩(wěn)定性, 大大提高蛋白質(zhì)產(chǎn)物水平。d:在構(gòu)建大腸桿菌表達(dá)載體時(shí)，通常是添加全部終止密碼子 , 阻止核糖體跳躍 (skipping )現(xiàn)象。e:大腸桿菌格外偏愛(ài)使用終止密碼子UAA, 當(dāng)其后連上一個(gè)U而形成四聯(lián)核苷酸的情況下 , 轉(zhuǎn)譯終止效率便會(huì)得到進(jìn)一步加強(qiáng)轉(zhuǎn)譯起始序列a: mRNA的5末端之獨(dú)特的結(jié)構(gòu)特征, 是決定mRNA轉(zhuǎn)譯起始效率的主要因素。b: 在構(gòu)建外源基因的高效表達(dá)載體時(shí) , 需認(rèn)真選擇有效的轉(zhuǎn)錄起始序列。c: 未鑒定出通用有效的轉(zhuǎn)譯起始序列的保守結(jié)構(gòu) (KOZAK SE

4、QUENCE)目的基因保護(hù)堿基內(nèi)切酶1內(nèi)切酶2保護(hù)堿基Gene cloningYour destination vectorR1R2Your linearized vectorX基因R1R2+1. 雙酶切目的基因和載體及切膠回收：附件目的基因保護(hù)堿基內(nèi)切酶1內(nèi)切酶2保護(hù)堿基2. 連接轉(zhuǎn)化：附件3. 雙酶切鑒定：附件4. 測(cè)序：附件1：DNA marker; 2：重組質(zhì)粒pET28-X雙酶切; 3：空質(zhì)粒pET28a雙酶切圖1.3 重組質(zhì)粒pET28-X雙酶切鑒定圖1.4 X基因發(fā)生點(diǎn)突變(AGUAGC)，但為同義突變（Ser ）Protein expression 檢測(cè)該重組質(zhì)粒在BL21中是

5、否能被誘導(dǎo)表達(dá)，并確定在不同IPTG濃度和時(shí)間蛋白誘導(dǎo)效率的差異。挑單克隆菌落于7ml含有相應(yīng)抗生素LB培養(yǎng)液的50ml培養(yǎng)管中，37C振蕩培養(yǎng)過(guò)夜（8-16h）。37C，1mM IPTG終濃度誘導(dǎo)蛋白。取700ul過(guò)夜搖菌加入7ml含有相應(yīng)抗生素LB培養(yǎng)液的50ml離心管中，于OD6000.4-0.6（1:100接種，37C培養(yǎng)時(shí)，細(xì)菌生長(zhǎng)至OD6000.4-0.6約需2h）時(shí)加入終濃度為1mM的IPTG。誘導(dǎo)前在37C培養(yǎng)的菌液中取1ml未誘導(dǎo)的對(duì)照，按1ml菌液OD600100l的比例(如1ml OD600為0.4的菌加40ul buffer)加入2上樣緩沖液，混勻，-20保存或直接上樣

6、。根據(jù)蛋白分子量大小配置相應(yīng)濃度的SDS膠。（50KD蛋白10或12）誘導(dǎo)3-4h后收集菌液，取1ml樣品，測(cè)OD600，10000rpm，1min離心去上清后加入相應(yīng)體積的2上樣緩沖液，vortex。將收集的菌液在水中煮5min，上樣10l ，120V電壓走濃縮膠，加電壓至150V進(jìn)入分離膠電泳，直到染料剛好走到膠底。取下膠，考馬斯亮藍(lán)染色至少30min（染液若是新配的，染20min就夠了），脫色液脫色。若急需看到結(jié)果，可以將膠在500ml蒸餾水中煮沸兩次即可看到條帶。IPTG inductionEven in the absence of IPTG, there is some expre

7、ssion of T7 RNA polymerase from the lacUV5 promoter.The degree of toxicity will vary from protein to protein. pET system manual, Novagen, 11th Edition1：蛋白marker；29：分別用IPTG 誘導(dǎo)表達(dá)0、1、 2、3、4、6、8、12 h。圖1.6 X蛋白表達(dá)條件：誘導(dǎo)時(shí)間優(yōu)化1：0mmol/L； 2：0.25mmol/L；3：0.5mmol/L；4：0.75mmol/L；5：1.0mmol/L； 6：1.5mmol/L；7：2.5mmol/L

8、； 8：3.5mmol/L； 9：5.0mmol/L。圖1.5 X蛋白表達(dá)條件：IPTG濃度優(yōu)化X蛋白在大腸桿菌中表達(dá)條件優(yōu)化Protein PurificationCharacterize function, activity, structureUse in assaysRaise antibodiesmany other reasons . Guidelines for protein purificationDefine objectivesDefine properties of target protein and critical contaminantsMinimize the

9、 number of stepsUse a different technique at each stepDevelop analytical assaysAdapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition ACProtein preparation, extraction, clarificationCell growth, protein over-expressionCell lysisRemoval of cell debrisFrom: Protein Puri

10、fication Handbook. Amersham Biosciences. 18-1132-29, Edition ACProtein isolation, concentration, and stabilizationReversible precipitation with salt or organic moleculesFrom: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition ACIntermediate PurificationLiquid chromatography (lo

11、wer resolution, lower cost)From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition ACAn introduction to liquid chromatographyProtein solution applied to a columnColumn = solid porous matrix (stationary phase) + liquid (mobile phase)Proteins separated based on differing interac

12、tions with stationary and mobile phasesMobile phase conditions can be adjusted to increase or decrease affinity of protein for stationary phase (gradient)Size exclusion chromatographyBigger ProteinsSmaller ProteinsAffinity ChromatographyAffinity ChromatographyMost commonly-used chromatography techni

13、queCan be used on protein with natural ligandsOften involves covalent attachment of affinity tag to protein Because of unique tag, provides rapid, specific cleanup in one chromatography step*Can allow for automation of protein purificationPopular Small Affinity TagsTagMatrixEluting AgentResiduesSequ

14、enceNotesHisNi2+-NTA, Talon, Cobaltimidazole or low pH5-15HHHHHnative or denaturing; inexpensive resinFLAGanti-FLAG antibodylow pH or EDTA/EGTA8DYKDDDDKhigh specificity; harsh elution cond.Strep IImodified streptavidindesthiobiotin8WSHPQFEKexpensive resinS-peptideS-proteinenzymatic cleavage15KETAAAK

15、FERHMDSdetection possible; expensive resinPopular Large Affinity TagsTagMatrixEluting AgentSizeNotesMBPamylosemaltose40 kDasolubility + secretion enhancer; inexpensive resinGSTglutathionereduced glutathione26 kDainexpensive resincellulose-binding domainscellulosewater or GdHCl4-20 kDasecretion enhan

16、cercalmodulin-binding peptidecalmodulinEDTA/EGTA4 kDadetection possible; expensive resinHis-patch thioredoxinNi2+-NTA, Talonimidazole11.7 kDasolubility + S-S enhancer鎳柱親和層析詳細(xì)步驟：附件1：空質(zhì)粒未誘導(dǎo)；2：空質(zhì)粒IPTG誘導(dǎo)；3：細(xì)菌裂解液上清（可溶部分）；4：細(xì)菌裂解液沉淀（不可溶部分）；(B) 切膠回收 1：蛋白marker ；2：重組質(zhì)粒未誘導(dǎo)；3：重組質(zhì)粒IPTG誘導(dǎo)；4：空白孔； 5：純化重組蛋白； (C) Ni

17、柱親和層析：1-8為依次的蛋白過(guò)柱洗脫液圖1.7 X蛋白的可溶性分析(A)和純化(B、C)蛋白純化結(jié)果1.親和層析柱2.切膠回收（附件）可溶性蛋白 or包涵體純化Polishing stepsFrom: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition ACLiquid chromatography (higher resolution, higher cost)Basic scheme of protein purificationLiquid chromatography (higher res

18、olution, higher cost)Liquid chromatography (lower resolution, lower cost)Reversible precipitation with salt or organic moleculesCell growth, protein over-expressionCell lysisRemoval of cell debrisFrom: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition ACTag RemovalNH2proteinta

19、glinkerDDDDKproteaseconsiderations:effect on structureeffect on functionflexibilityprotein 1 sequence Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins Protein Expression and PurificationVolume 48, Issue 1, July 2006, Pages 1-13 Protein dete

20、ction methodsSDSVisual confirmationWestern blotUV SpectrophotometryAbsorbance 280 nmDue mostly to TrpProtein calculated with Beers LawColorimetric TechniquesColor change proportional to proteinBradford, Lowry, BCAJ.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.2

21、91：未加IPTG誘導(dǎo)的重組質(zhì)粒；2：IPTG 誘導(dǎo)的重組質(zhì)粒；3：空白孔；4： 純化后的重組蛋白圖1.8 重組蛋白的Western blot 分析26kDa1243Anti-6His antibodyFinal steps in purificationCheck purity by detection methodsConcentrate your proteinPrecipitationCentriconsSmall column with high binding capacityChoose a storage buffer and storage conditionsConside

22、r intended use of proteinStabilizing additivesFlash freeze protein and store at -80o CConfirm identity of purified proteinMass spectrometrysequencingAnalytical assaysHelpful references and guides附件：Protein_Expression_Protocol_3th.docpET system manual, Novagen, 11th EditionAmersham Biosciences “Prote

23、in purification handbook.” 18-1132-29, Edition AC. Go to following URL and download pdf of Protein Purification Handbook:Affinity Purification: Arnau, J., Lauritzen, C., Petersen, G.E., Pedersen, J. Prot. Expr. Purif. 48 (2006) 1-13.J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29Part 2：抗體制備、應(yīng)用單抗制備多抗制備：附件抗體鑒定效價(jià)特異性Principle of McAb Production抗原經(jīng)FCA或FIA充分乳化后免疫家兔。首次免疫佐劑用FCA，第二、三次用FIA。家兔脊柱兩旁選5點(diǎn)皮下注射，每點(diǎn)注攝0.2ml， 間隔后2周選不同點(diǎn)注射，每次免疫的抗原量約 350g/兔。第三次免疫后10天，耳動(dòng)脈取血檢測(cè)抗體效價(jià)。雙相瓊脂擴(kuò)散實(shí)驗(yàn)效價(jià)至少達(dá)到1:16；ELISA效 價(jià)達(dá)到105即可放血。最后一次取血采用頸動(dòng)脈放血。新西蘭兔（附件）圖1.9 兔多克隆抗體效價(jià)測(cè)定-瓊脂糖凝膠免疫