Linker mutagenesis of the Caulobacter crescentus S-layer protein: Toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion
Linker mutagenesis of the Caulobacter crescentus S-layer protein: Toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion
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DOI：
10.1128/jb.179.3.601-611.1997
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发表时间：
1997-02-01
影响因子：
3.2
通讯作者：
Smit, J
中科院分区：
文献类型：
--
作者：
Bingle, WH;Nomellini, JF;Smit, J
利用连接子插入诱变技术对革兰氏阴性菌新月形Caulobacter crescentus的副晶表面层（S-layer）蛋白（RsaA）进行修饰，在克隆的RsaA基因中鉴定出11个独特的BamHI连接子插入；在蛋白质水平上，这些连接子插入在1026个氨基酸的RsaA蛋白的极N端到极C端引入了4到6个氨基酸；在RsaA N端插入yl连接肽导致分泌蛋白脱落到生长培养基中，表明RsaA N端参与细胞表面锚定。一个连接肽插入RsaA C端（氨基酸784）对s层生物发生没有影响，而另一个连接肽插入（氨基酸907）破坏了蛋白质的分泌，这表明RsaA在氨基酸784的C端有一个分泌信号，靠近或包含氨基酸907。与极端的N端或C端连接肽插入不同，位于RsaA初级序列中心的连接肽插入对s层生物发生没有明显影响。利用新引入的连接子编码限制性位点，编码s层蛋白最后242个c端氨基酸的rsaA基因3′片段在异源大肠杆菌lacZ转录和翻译起始信息中得到表达，该rsaA c端部分被分泌到生长培养基中，证实了c端分泌信号的存在。使用RsaA C末端的分泌外源蛋白在C crescentus被融合了109个氨基酸的传染性造血坏死病毒包膜糖蛋白,病原体的salmonid鱼,到最后RsaA C端242个氨基酸,由此产生的混合蛋白质被成功地分泌生长介质和占总蛋白的10%在固定相文化,基于这些结果和RsaA主要序列的特性,我们认为弯月牙菌s层蛋白是由I型分泌系统分泌的，依赖于稳定的c端分泌信号，类似于大肠杆菌α溶血素，这是该途径分泌s层蛋白的第一个例子。
Linker insertion mutagenesis was used to modify the paracrystalline surface layer (S-layer) protein (RsaA) of the gram-negative bacterium Caulobacter crescentus, Eleven unique BamHI linker insertions in the cloned rsaA gene were identified; at the protein level, these linker insertions introduced 4 to 6 amino acids at positions ranging from the extreme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein,;yl linker peptide insertions in the RsaA N terminus caused the secreted protein to be shed into the growth medium, suggesting that the RsaA N terminus is involved in cell surface anchoring. One linker-peptide insertion in the RsaA C terminus (amino acid 784) had no effect on S-layer biogenesis, while another (amino acid 907) disrupted secretion of the protein, suggesting that RsaA possesses a secretion signal lying C terminal to amino acid 784, near or including amino acid 907, Unlike extreme N- or C-terminal linker-peptide insertions, those more centrally located in the RsaA primary sequence had no apparent effect on S-layer biogenesis. By using a newly introduced linker-encoded restriction site, a 3' fragment of the rsaA gene encoding the last 242 C-terminal amino acids of the S-layer protein was expressed in C. crescentus from heterologous Escherichia coli lacZ transcription and translation initiation information, This C-terminal portion of RsaA was secreted into the growth medium, confirming the presence of a C-terminal secretion signal, The use of the RsaA C terminus for the secretion of heterologous proteins in C. crescentus was explored by fusing 109 amino acids of an envelope glycoprotein from infectious hematopoietic necrosis virus, a pathogen of salmonid fish, to the last 242 amino acids of the RsaA C terminus, The resulting hybrid protein was successfully secreted into the growth medium and accounted for 10% of total protein in a stationary-phase culture, Based on these results and features of the RsaA primary sequence, we propose that the C. crescentus S-layer protein is secreted by a type I secretion system, relying on a stable C-terminal secretion signal in a manner analogous to E. coli alpha-hemolysin, the first example of an S-layer protein secreted by such a pathway.