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1、基因原核表达系统,转录载体,翻译载体,LIC(连接非依赖的克隆),图3.5 SCMRP基因原核表达M:蛋白质分子量标准;泳道1:IPTG诱导BL21(DE3 pET-32b)总蛋白;泳道24:IPTG诱导BL21(DE3 pET-32bS)总蛋白。Fig.3.5 Prokaryotic expression of gene SCMRPM:Protein Molecular Weight Marker;lane1:the whole cell protein of BL21(DE3 pET-32b)induced by IPTG;lane 24:the whole cell protein of
2、 BL21(DE3 pET-32bS)induced by IPTG.,图3.6 SCMRP融合蛋白大肠杆菌中表达的SDS-PAGE电泳图M:蛋白质分子量标准;1、4、7泳道分别是IPTG诱导pET32b表达2、4、6小时的DE3的总蛋白;2、5、8泳道分别是IPTG诱导重组质粒pET32bS表达2、4、6小时的DE3的总蛋白;3、6、9泳道分别是无IPTG诱导时,重组质粒pET32bS表达2、4、6小时的DE3的总蛋白。Fig.3.6 SDS-PAGE result of SCMRP recombinant protein in E.coli strain DE3.The results s
3、howed the fused proteins are expressed in E.coli by IPTG induction.The fused protein has a molecular mass of about 20KD.The samples were collected 2h,4h,6h after induction by IPTG from pET32b(lane1,4,7)and from pET32b-SCMRP(lane2,5,8),respectively.The samples were collected 2h,4h,6h from pET32b-SCMR
4、P(lane3,6,9),respectively.M:Protein Molecular Weight Marker.,图3.7 SCMRP基因原核表达产物溶解形式分析M:蛋白质分子量标准;泳道1、2:BL21(DE3 pET-32bS)表达蛋白质的可溶组分;泳道3:BL21(DE3 pET-32b)表达蛋白质的可溶组分;泳道5、6、7:BL21(DE3 pET-32bS)表达蛋白质的不溶组分;泳道8:BL21(DE3 pET-32b)表达蛋白质的不溶组分。Fig.3.7 The dissolvability analysis of prokaryotic expression produc
5、t of the gene SCMRPM:protein marker;lane1-2:dissolvable group of fusion protein by BL21(DE3 pET-32bS);lane3:dissolvable group of fusion protein by BL21(DE3 pET-32b);lane5-7:insolvable group of fusion protein by BL21(DE3 pET-32bS);lane8:insolvable group of fusion protein by BL21(DE3 pET-32b).,图3.8 SC
6、MRP原核表达产物Western blot分析M:蛋白质分子量标准;泳道1:Ni-NTA纯化SCMRP;泳道2:IPTG诱导BL21(DE3 pET-32b)总蛋白;泳道3:IPTG诱导BL21(DE3 pET-32bS)总蛋白;泳道4:无IPTG诱导BL21(DE3 pET-32bS)总蛋白。Fig.3.8 Western blot analysis of prokaryotic expression product of SCMRPM:protein marker;lane1:purification protein through Ni-NTA;lane2:the whole cell
7、protein expressed by BL21(DE3 pET-32b)induced by IPTG;lane3:the whole cell protein expressed by BL21(DE3 pET-32bS)induced by IPTG;lane4:the whole cell protein expressed by BL21(DE3 pET-32bS)without IPTG induction.,蛋白质溶解方式调节,与溶解性高的蛋白质序列融合如GST、Trx(硫氧还蛋白)、NusA与催化二硫键形成的酶融合如Trx、DsbA、DsbC与信号肽融合DsbA、DsbC低温
8、培养、诱导,凝血酶,肠激酶,原核表达宿主菌的选择,BL21(DE3):为 DE3 溶原菌,其染色体上带有一拷贝由 lacUV5 启动子控制的 T7 RNA 聚合酶基因。命名为 pLysS 和 pLysE 的宿主菌带有编码 T7 溶菌酶(为 T7 RNA 聚合酶的天然抑制物)的 pET 相容性质粒。带有 pLysS 的细胞产生少量溶菌酶,而 pLysE 宿主菌产生更大量酶。这些菌株用于在诱导前抑制 T7 RNA 聚合酶的基础表达,这样可以稳定编码影响细胞生长和活力的目标蛋白的 pET 重组体。,Rosetta(DE3):Rosetta 宿主菌从 BL21 衍生而来,可增强带有大肠杆菌稀有密码子的真核蛋白的表达。该菌株通过一个相容性氯霉素抗性质粒补充密码子 AUA、AGG、AGA、CUA、CCC 和 GGA 的 tRNAs。这样 Rosetta 菌株提供了“万能”的翻译,从而避免因大肠杆菌密码子使用频率导致的表达限制。,