pet系統(tǒng)原核表達詳細總結

1、1會計學pet系統(tǒng)原核表達詳細總結系統(tǒng)原核表達詳細總結不依賴連接反應的克隆方法（不依賴連接反應的克隆方法（LIC）nThe 35 exonuclease activity of T4 DNA polymerase removes nucleotides until it encounters a residue corresponding to the single dNTPpresent in the reaction mix. At this point the 53 polymerase activity of the enzyme counteractsthe exonuclease

2、activity to effectively prevent further excision.npET載體最初是由Studier及其同事構建。 (Studier and Moffatt,1986; Rosenberg et al., 1987; Studier et al., 1990). 包括兩種載體轉錄載體（用于表達本身含有核糖體結合位點和起始密碼子目的基因）和翻譯載體（載體上帶有核糖體結合位點）。一般來說，轉錄載體用于表達來自原核的基因，而翻譯載體表達真核基因。nBL21(DE3) 基因型： F ompT hsdSB (rB mB) gal dcm (DE3) 無抗性n重組質粒轉化到

3、帶有T7RNA聚合酶基因 的大腸桿菌中，即可開始產生蛋白了。這些菌株都是DE3溶原菌。噬菌體DE3是一種衍生噬菌體，帶有噬菌體21抗性區(qū)和lacI基因，lacUV5啟動子，以及T7RNA聚合酶基因。這一區(qū)段被插入int基因，因此阻止了DE3在沒有輔助噬菌體時整合到染色體上或從染色體切出，一旦形成DE3溶原狀態(tài)，就只有受IPTG誘導的lacUV5啟動子指導T7RNA聚合酶基因轉錄。 cAMP/CRP的刺激并不敏感。然而，最近研究發(fā)現(xiàn)rDE3宿主菌在缺少葡萄糖的培養(yǎng)基生長到穩(wěn)定期時，cAMP同樣對lacUV5產生了去阻遏作用。盡管宿主菌長到穩(wěn)定期并不推薦，菌體培養(yǎng)過夜（16h）后轉入含有1%的葡萄

4、糖的培養(yǎng)基中，可以有效避免去阻遏作用，因為glucose可以cAMP的產生。pLysS (or pLysE）有助于目的蛋白的抽提）有助于目的蛋白的抽提nThe presence of pLysS (or pLysE) has the further advantage of facilitating the preparation of cell extracts. After the target protein has accumulated, the cells are collected and suspended in a buffer such as 50 mM Tris-HCl,

5、 2 mM EDTA, pH 8.0. Simply freezing and thawing, or adding 0.1% Triton X-100, will allow the resident T7 lysozyme to efficiently lyse the cells. This property can make it advantageous to carry pLysS in the cell even when it is not required for stabilizing the target plasmid.n Prepare pET Vector Prep

6、are Insert DNA Clone Insert into pET Vector Transform into Expression Host Induce and Optimize Expression of Target Protein Scale-up Purify Target ProteinnFor a standard reaction using DNA fragmentswith 24 base sticky ends, use 50-100 ng(0.0150.03 pmol) of pET vector with 0.2 pmol insert (50 ng of a

7、 500 bp fragment) in avolume of 20l.Assemble the following components in a 1.5 ml tube (these components are available separately in the DNA Ligation Kit, Cat. No. 69838-3). Add the ligase last.12,000 g離心1min.徹底去除上清.3.用100ul solutionI懸浮菌體. 4.加200ul新鮮配制的0.2 N NaOH, 1% SDS，顛倒混勻，冰上放置3min.5.加150ul150 l

8、of ice-cold 3 M NaOAc, pH 5.2。顛倒混勻，冰上放置5min。6.12000rpm，離心5min。轉移上清到一個新的Ep管。7.加400 l TE-buffered phenol:chloroform:isoamyl alcoho(25:24:1)，渦旋震蕩30s。8.12,000 g for 5 min at 4C.9.倒掉上清，加400ul乙醇。10.重懸沉淀，30ul含20ug/mlRNAse的TEbuffer。37放置15min。 solutionI（ice-cold 50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mMEDT

9、A預冷）nFor pET constructions carrying the “plain” T7 promoter, a final concentration of 0.4 mM IPTG is recommended, while 1 mM IPTG is recommended with vectors having the T7lac promoter. An準備誘導表達準備誘導表達nPick a single colony from a freshly streaked plate and inoculate 50 ml LB containing the appropriate

10、 antibiotic in a 250 ml Erlenmeyer flask. (For good aeration, add medium up to only 20% of the total flask volume.)nAlternatively, inoculate a single colony or a few microliters from a glycerol stock into 2 ml LB medium containing the appropriate antibiotic. Incubate with shaking at 37C until the OD

11、600 reaches 0.61.0. Store the culture at 4C overnight. The following morning, collect the cells by centrifugation (30 sec in a microcentrifuge). Resuspend the cells in 2 ml fresh medium plus antibiotic and use this to inoculate 50 ml medium.IPTG將阻止任何帶有T7RNA聚合酶誘導基因和功能目的質粒的細胞形成菌落。III，IV不適用于pLysS，pLysE

12、質粒和T7lac啟動子。 The optimal scheme and time course for induction can vary, because the characteristics of each target gene product are unique.Example：37生長常常會導致一些蛋白質積累形成包涵體，而30生長則可能產生有活性的蛋白。如果想利用一些pET載體中的信號肽序列輸出蛋白的話，在25或30生長和培養(yǎng)可能是最優(yōu)化的。在某些情況下，低溫（15-20）延長誘導時間（過夜）可以使溶解性蛋白產量達到最大。近些年來，包涵體的解折疊的方法學上取得了很大的進步。報道

13、了很多解折疊方案，不同的目的蛋白的解折疊方案不同，主要是根據經驗來決定。蛋白質解折疊試劑盒提供了一套試劑，能很方便的促進一些蛋白的重折疊。pET-32, pET-39 and pET-40n溶解性可以通過選擇載體，克隆位點或是宿主菌來調控。例如，pET-32系列載體產生硫氧還蛋白的融合蛋白，可以增加胞質中可溶蛋白的產量。AD494或BL21trxB菌株可以在胞質中形成二硫鍵。nIf your target protein contains one or more essential disulfide bonds, the combination of a pET-32 vector and

14、a trxBhost may prove to be optimal because disulfide bond formation in the cytoplasm appears to bedependent on the presence of thioredoxins (Stewart et al., 1998).與胞質中不同，大腸桿菌細胞周質是一個氧化的環(huán)境，含有催化二硫鍵形成的酶。要想得到正確折疊，有活性的含二硫鍵的目的蛋白，一個常用的策略就是，將異源蛋白運輸?shù)郊毎苜|中。一般來說，通過目的基因與號肽基因融合，可以使表達蛋白定位于周質。注意：過表達的DsbC酶是以氧化形式存在的，

15、必須暴露在還原試劑中（0.1-1.0mMDTT）在體外才表現(xiàn)出二硫鍵異構酶的活性。Typically, a DsbC fusion protein expressed from pET-40b(+) is first purified by HisBind chromatography. Prior to exposing the fusion protein to a reducing agent, either EDTA should be added to a final concentration of 1 mM, or the sample should be dialyzed to

16、 remove residual Ni2+.nAnother factor that appears to influence target protein stability is the amino acid immediately following the N-terminal methionine (penultimate amino acid). The amino acid at this position determines the removal of N-terminal fMet. Processing is catalyzed by methionyl aminope

17、ptidase and is governed by the following relationship: the degree of removal decreases as the size of the penultimate amino acid side chain increases (Hirel et al., 1989; Lathop et al., 1992). In practice, little or no processing was observed by these authors when the following amino acids occupied

18、the penultimate position: His, Gln, Glu, Phe, Met, Lys, Tyr, Trp, Arg. Processing ranged from 16%97% when the remaining amino acids occupied this position. Tobias et al. (1991) have determined the relationship between a proteins amino terminal amino acid and its stability in bacteria, i.e., the N-en

19、d rule. They reported protein half-lives of only 2 minutes when the following amino acids were present at the amino terminus: Arg, Lys, Phe, Leu, Trp, and Tyr. In contrast, all other amino acids conferred half-lives of 10 hours when present at the amino terminus in the protein examined.Leu in the penultimate position would