In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.
In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.
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DOI：
10.1371/journal.pone.0127504
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Arpino JA;Baldwin AJ;McGarrity AR;Tippmann EM;Jones DD
扩展遗传密码方法是一种强大的手段，可以在确定的残基位置为蛋白质添加新的和有用的化学物质。其中一个用途是引入非生物反应性化学手柄，用于特定位点的蛋白质生物相容性正交偶联。由于我们目前对非规范氨基酸（nAAs）对蛋白质结构-功能关系的影响的信息有限，合理的蛋白质工程是选择合适位点的“命中和错过”方法。此外，教条建议表面暴露的天然残留物应该是引入新的偶联化学的主要焦点。在这里，我们描述了一种定向进化方法来引入和选择框架内密码子替换，以促进具有nAAs的工程蛋白。为了证明这种方法，将通常重编程的琥珀色停止密码子（TAG）随机引入到两种不同的蛋白质中：生物纳米技术上重要的cytb562和治疗蛋白KGF。目标蛋白在基因水平上通过TEV蛋白酶位点与sfGFP连接。在没有nAA的情况下，框架内的TAG将终止翻译，导致非荧光细胞表型。在nAA存在的情况下，TAG会编码nAA结合，从而在大肠杆菌上注入绿色荧光表型。内源性表达的TEV蛋白酶的存在将体内靶蛋白从其融合到sfGFP中分离出来，如果作为可溶性融合产物表达的话。利用这种方法，我们结合了叠氮化物反应手柄，并通过菌株促进叠氮化物-炔环加成（SPAAC）鉴定了可与荧光染料偶联的残基位置。有趣的是，通过SPAAC进行有效偶联的最佳位置是通过分析其确定的三维结构而将天然侧链掩埋的残基，因此可能不是通过合理的蛋白质工程选择的。分子模拟表明，这些埋藏的天然残基可能部分暴露于含有nAA的叠氮化物取代上。
Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a “hit and miss” approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b 562 and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on substitution to the azide containing nAA.
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影响因子：
14.9
作者：
Edwards WR;Busse K;Allemann RK;Jones DD
通讯作者：
Jones DD
影响因子：
10.8
作者：
Della Pia, Eduardo Antonio;Chi, Qijin;Elliott, Martin
通讯作者：
Elliott, Martin
DOI：
10.1073/pnas.0707090104
发表时间：
2007-10-23
影响因子：
11.1
作者：
Baskin, Jeremy M.;Prescher, Jennifer A.;Bertozzi, Carolyn R.
通讯作者：
Bertozzi, Carolyn R.
影响因子：
4.8
作者：
Antonczak, Alicja K.;Simova, Zuzana;Tippmann, Eric M.
通讯作者：
Tippmann, Eric M.
影响因子：
14.9
作者：
Baldwin AJ;Busse K;Simm AM;Jones DD
通讯作者：
Jones DD