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1、第七章 外源基因表达n一、表达机制n1起始转录：转录起始的速率是gene表达的限速步骤，用诱导型启动子（Lac、Trp、 PL/PR、 tac、组成型启动子：T7）n2MRNA延伸与稳定：正常终止序列、减少DNA反向转录产生反义mRNA、polyA掺入对3正确加工n3MRNA有效翻译：AUG首选起始codon、SD序列与16srRNA互补结合，含有至少AGGAGG中4个碱基、SD与ATG间距离3-9bp、翻译起始区周围不形成明显二级结构、codon选择性相近（没有明显偏好性不同）n4Pr稳定性：构建融合pr、分泌pr、包涵体pr、pr水解酶缺陷型受体细胞第七章 基因表达系统n外源基因表达系统n
2、泛指目的基因与表达载体重组后，导入合适受体细胞，并在其中有效表达，产生目的基因产物n1E. coli表达系统：n常用lac & tac：以lac操纵子调控机制为基础：lacP lacO 结构基因（lacZ, Y, A），lacI 阻遏pr，IPTG诱导，nPL & PR：以phage溶原/溶菌循环nT7启动子：T7RNA聚合酶识别序列在pET中MCS上端，RNA聚合酶位于BL21(DE3)基因组中，上端为lac启动子调控，IPTG第七章 基因表达系统n外源pr表达形式：n包涵体pr：n融合pr：n寡聚型pr：多表达单元重组、多顺反子、多编码序列The expression vectorMost
3、 important elements in the vectoruPromoter F-35: TTGACA (E. coli )F-10: TATAAT (E. coli )uTerminator (hairpin)uRibosome binding site (RBS)uSelection markeruVector replication sequence (ori)uPolylinker (MCS)In the gene/vectoruStart (ATG)uStop (1-2 TAA, (TAG, TGA)uSignal peptidetac promoterHybrid prom
4、oter fusion from lac and trp promoters+ Combines strong RNA polymerase binding sequences+ 5 x stronger than lac promoter+ 20-30% of total protein+ Induced by IPTG lPL promoterIsolated from l bacteriophage - repressor (cI) binds to lPL promoter+/- cI 857 variant heat inducible 30 degrees, expression+
5、/-Alternatively use special strains with repressor under control of trp promoter which is induced by Trp addition+Tightly regulated - good for toxic proteins Target protein Target genelPLRepressor genecI repressortrp promoterTrp+T7 system (pET-system)nT7 RNA polymerase gene controlled by lac promote
6、rnIPTG induces T7 RNA polymerase productionnT7 RNA polymerase transcribes gene controlled by T7 promoternTarget gene can not be activated by host RNA polymerasenSpecial strain needednlacO in pET and pLysS reduces leakinessGAL systemGene cloned under control ofthe GAL4 promoterInduction by galactose as thesole carbon sourceStronger induction by PGAL1Gal4p transcriptional activator- 1-5% of total protein- Leaky promoter赵子昂
