A structurally informed autotransporter platform for efficient heterologous protein secretion and display.
A structurally informed autotransporter platform for efficient heterologous protein secretion and display.
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DOI：
10.1186/1475-2859-11-85
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发表时间：
2012-06-18
影响因子：
6.4
通讯作者：
Luirink J
中科院分区：
文献类型：
--
作者：
Jong WS;Soprova Z;de Punder K;ten Hagen-Jongman CM;Wagner S;Wickström D;de Gier JW;Andersen P;van der Wel NN;Luirink J
自给自足的自转运蛋白（AT）途径，普遍存在于革兰氏阴性菌，结合了一个相对简单的蛋白分泌机制与高运输能力。AT由分泌的乘客结构域和促进乘客穿过细胞包膜转移的β结构域组成。它们具有很大的潜力，用于重组蛋白的细胞外表达，但它们的开发受到载体AT的有限结构知识的影响。利用其晶体结构，我们已经将大肠杆菌AT血红蛋白蛋白酶（Hbp）改造成用于分泌和表面展示异源蛋白的平台，使用结核分枝杆菌疫苗靶ESAT 6作为模型蛋白。基于Hbp晶体结构，选择五个乘客侧结构域，并一个接一个地被ESAT 6替换，而β-螺旋核心结构（β-茎）保持完整。重组Hbp-ESAT6嵌合体可稳定分泌到大肠杆菌培养基中。杆菌另一方面，含有截短的β-茎的Hbp-ESAT 6融合体在易位后表现出不稳定性，证明了完整的β-茎的重要性。通过中断过客和β结构域之间的切割位点，构建了Hbp-ESAT 6展示变体，其保持细胞缔合并促进ESAT 6的有效表面暴露，如通过蛋白酶K可及性和全细胞免疫EM分析所判断的。在替换替代AT的乘客侧结构域EspC后，ESAT 6也被有效分泌，表明该方法更普遍适用于AT。此外，Hbp-ESAT 6在单个编码基因拷贝的染色体整合后在减毒鼠伤寒沙门氏菌菌株中有效展示，证明了Hbp平台用于活疫苗开发的潜力。我们开发了第一个结构上知情的AT平台，用于高效分泌和表面展示异源蛋白。该平台具有开发重组活疫苗的潜力，并可用于需要细菌高水平分泌或展示重组蛋白的其他生物技术应用。
The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.
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影响因子：
15.3
作者：
Otto, B R;van Dooren, S J;Nuijens, J H;Luirink, J;Oudega, B
通讯作者：
Oudega, B
影响因子：
--
作者：
Kang, HY;Curtiss, R
通讯作者：
Curtiss, R
影响因子：
3.6
作者：
Jong, Wouter S. P.;ten Hagen-Jongman, Corinne M.;Luirink, Joen
通讯作者：
Luirink, Joen
DOI：
10.1073/pnas.1009491107
发表时间：
2010-10-12
影响因子：
11.1
作者：
Peterson, Janine H.;Tian, Pu;Bernstein, Harris D.
通讯作者：
Bernstein, Harris D.
影响因子：
5.6
作者：
Khan, Shekeb;Mian, Hira S.;Sandercock, Linda E.;Chirgadze, Nickolay Y.;Pai, Emil F.
通讯作者：
Pai, Emil F.