Recombining low homology, functionally rich regions of bacterial subtilisins by combinatorial fragment exchange.
Recombining low homology, functionally rich regions of bacterial subtilisins by combinatorial fragment exchange.
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DOI：
10.1371/journal.pone.0024319
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发表时间：
2011
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：
Jones DD
利用组合片段交换重组芽孢杆菌枯草杆菌蛋白酶的关键结构和功能的低同源性区域，以产生具有改变的底物谱的新的活性杂合蛋白酶。最多六个不同的区域，主要包括来自商业上重要的枯草杆菌蛋白酶Savinase的环残基，与六个其他枯草杆菌蛋白酶的结构等同区域交换。另外六种枯草杆菌蛋白酶来自不同的来源，包括嗜热和细胞内枯草杆菌蛋白酶以及其他学术和商业相关的枯草杆菌蛋白酶。Savinase在很大程度上耐受片段交换;用6个供体枯草杆菌蛋白酶序列中的5个合理替换所有6个区域保留了活性，尽管与Savinase相比降低了活性。使用组合方法来产生杂合Savinase变体，其中将源自每个区域的所有七种枯草杆菌蛋白酶的序列重组以产生新的区域组合。与Savinase和合理片段交换变体相比，产生了具有不同底物谱且具有更大表观活性的变体，其底物谱取决于每个区域的序列组合。
Combinatorial fragment exchange was utilised to recombine key structural and functional low homology regions of bacilli subtilisins to generate new active hybrid proteases with altered substrate profiles. Up to six different regions comprising mostly of loop residues from the commercially important subtilisin Savinase were exchanged with the structurally equivalent regions of six other subtilisins. The six additional subtilisins derive from diverse origins and included thermophilic and intracellular subtilisins as well as other academically and commercially relevant subtilisins. Savinase was largely tolerant to fragment exchange; rational replacement of all six regions with 5 of 6 donating subtilisin sequences preserved activity, albeit reduced compared to Savinase. A combinatorial approach was used to generate hybrid Savinase variants in which the sequences derived from all seven subtilisins at each region were recombined to generate new region combinations. Variants with different substrate profiles and with greater apparent activity compared to Savinase and the rational fragment exchange variants were generated with the substrate profile exhibited by variants dependent on the sequence combination at each region.
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影响因子：
5.6
作者：
Bershtein, Shimon;Goldin, Korina;Tawfik, Dan S.
通讯作者：
Tawfik, Dan S.
影响因子：
56.9
作者：
CARTER, P;WELLS, JA
通讯作者：
WELLS, JA
DOI：
10.1093/protein/2.4.271
发表时间：
1988-10-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
NEIDHART, DJ;PETSKO, GA
通讯作者：
PETSKO, GA
影响因子：
64.8
作者：
CARTER, P;WELLS, JA
通讯作者：
WELLS, JA
影响因子：
3.4
作者：
Lee, AY;Park, SG;Park, BC
通讯作者：
Park, BC