1、Recombinant protein secretion in Escherichia coli,汇报人：夏媛媛,IF: 9.5992005,分泌物Most bacteria secrete proteins such as degradative enzymes, toxins, and other pathogenicity factors into the extracellular environment. 分泌类型The type I, II, III, IV, and V secretion pathways are widespread among Gram-negative
2、bacteria and their mechanisms differ significantly. This review discusses the type I and type II mechanisms that are used most commonly for recombinant protein secretion in Escherichia coli K-12 or B strains. 渗漏表达Finally, protein secretion to the culture medium may also occur by leakage of periplasm
3、ic contents, and thus is not always mediated by specific transport mechanisms as will be discussed in this review.,1. Introduction,2. Recombinant protein secretion,Advantages,1. Simplified downstream processing 方便纯化 2. Enhanced biological activity 正确折叠 3. Higher product stability and solubility 防止聚集
4、 4. N-terminal authenticity of the expressed peptide N端正确 5. Increase the stability of cloned gene products 促进稳定性,Problems,incomplete translocation across the inner membrane 难跨内膜 insufficient capacity of the export machinery 转运不足 proteolytic degradation 蛋白酶降解,Advices,蛋白大小 Protein size may influence
5、secretion efficiency and that large cytoplasmic proteins may be physically impossible to translocate,信号肽及目的蛋白 The amino acid composition of the leader peptide and of the target protein is also important.,最优翻译速率There is an optimum rate of translation to achieve high-level secretion of heterologous pr
6、oteins, and secretion may drop off severely at higher rates.,2.1Type I secretion mechanism,2.2Type II secretion mechanism,Cytoplasmic membrane translocation,Extracellular secretion,How are recombinant proteins really transported?,2.1 Type I secretion mechanism,一步跨两层膜，大肠杆菌常用此途 径分泌高分子量的毒素和胞外酶。,Example
7、s of the secretion of recombinant proteins expressed as fusions to the HlyA signal sequence,2. 大肠杆菌中a溶血素 (HlyA)的一型转运方式最常见。,3. 504000 amino acids,Drawbacks,1. 分泌时信号肽不会被切除，要获得完整目的蛋白需切除信号肽。,2. 目的蛋白与大肠杆菌自身蛋白竞争，分泌量少,2.2 Type II secretion mechanism,有三条途径：the SecB-dependentPathway SecB依赖途径the signal recogn
8、ition particle (SRP) pathway 信号识别颗粒 (SRP)途径the twin-arginine translocation (TAT)pathway 双精氨酸转移(TAT)途径,Cytoplasmic membrane translocation 细胞质膜转移,Extracellular secretion 胞外分泌,the SecB-dependent Pathway SecB依赖途径,触发因子(TF)与初始肽链结合,TF一直结合着，防止与SRP结合 ，直到前体蛋白离开核糖体,蛋白被SecB识别 并引导蛋白到SecA处,SecB依赖途径,在质子驱动力下促成转运,信号
9、肽：1830AA，N端带正电，中间疏水核心，C端极性裂口 缺陷: 大肠杆菌转运能力有限 方法：提高SecA表达量优化SecA和SecB比例 问题：SecA的表达受SecA-mRNA结合复合物及上游基因secM调控,此策略需要设计疏水信号肽。 在细胞质中，若蛋白在SecB途径下折叠太快，此途径则更有优势,the signal recognition particle (SRP) pathway 信号识别颗粒 (SRP)途径,SRP recognises its substrates by the presence of a hydrophobic signal sequence,SRP识别,SR
10、P核糖体复合体与FtsY作用，释放肽链到转运处,信号肽结构：与Sec类似，大约38AA， N端保守序列S/T-R-R-X-F-L-K 优点：穿越内膜时已是成熟蛋白 缺点：与Sec途径相比，效率低，时间长，分泌蛋白不大于142KDa需要共表达tatABC，消耗ATP，转运能力有限,the twin-arginine translocation (TAT)pathway 双精氨酸转移(TAT)途径,This pathway has been termed the TAT system because preproteins transported by it contain two consecu
11、tive and highly conserved arginine residues in their leader peptides.,在细胞质中折叠,与辅因子结合,信号肽被TatC识别,由TatEA形成的通道释放,Extracellular secretion 胞外分泌,Advantages,通过II型途径分泌蛋白到胞外构成了主要终端分支(MTB),这一过程需要Secreton。 大肠杆菌基因组中有MTB元件基因，但在实验条件下不表达。,方法：过量表达分泌元件优化培养条件促进大肠杆菌自己表达元件,周质渗漏可行性,方法,How are recombinant proteins really
12、 transported？,A novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli,天然分泌蛋白,xylanase 木聚糖酶 a-amylase 淀粉酶,天然胞内蛋白,xylose isomerase 木糖异构酶 trehalose synthase 海藻糖合成酶,xylanase 木聚糖酶,与角质酶共表达后 培养时间缩短一半 产量是单独分泌的7.9倍,pETDuet-cutinase-pelB-xynA,pETDuet-pelB-xynA,(1)pET
13、Duet-pelB-xynA 胞外部分; (2)pETDuet-cutinase-pelB-xynA 胞外部分; (3)pETDuet-pelB-xynA 胞内破碎上清; (4)pETDuet-cutinase-pelB-xynA 胞内破碎上清; (5)pETDuet-pelB-xynA 胞内破碎沉淀; (6) pETDuet-cutinase-pelB-xynA 胞内破碎沉淀,pETDuet-cutinase-xynA.目的蛋白无信号肽时，无论胞内胞外均无酶活，且都形成了包涵体。,分泌与细胞裂解无关。,a-amylase 淀粉酶,pETDuet-pelB-amy,pETDuet-cutina
14、se-pelB-amy,pETDuet-pelB-amy 胞外部分 pETDuet-cutinase-pelB-amy 胞外部分,最终酶活无明显变化，但达到最大酶活的时间缩短了,目的蛋白无信号肽时会形成包涵体。,xylose isomerase 木糖异构酶,pETDuet-xylA 胞外部分 pETDuet-cutinase-xylA 胞外部分,多于一半目的蛋白分泌出去 细胞未裂解,pETDuet-tres 胞外部分 pETDuet-cutinase-tres 胞外部分,trehalose synthase 海藻糖合成酶,胞内蛋白分泌影响因素 分子量：265KDa 表达量：胞内蛋白大量表达,谢谢,
