SfiNX: a method for assembly of protein coding sequences with high success rates
SfiNX: a method for assembly of protein coding sequences with high success rates
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SfiNX：一种高成功率的蛋白质编码序列组装方法
DOI：
10.1007/s10529-016-2042-2
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发表时间：
2016
影响因子：
2.7
通讯作者：
Keigo HISAMATSU
中科院分区：
文献类型：
--
作者：
Kazuyuki TAKAI;Keigo HISAMATSU
目的将两个NdeI-XhoI基因片段连接到带有AnSfiI酶切位点的载体上，以评价构建可在大肠杆菌中表达的多顺反子基因的成功率。将插入在pET-22b的NdeI和XhoI位点之间的3个小麦eEF1B亚基基因转移到含有壮观霉素抗性基因的SfiI载体中。然后，用氨苄西林抗性基因替换产生的质粒中的标记基因。这些质粒用于通过含有核糖体结合位点的接头寡核苷酸连接两个不同的基因。在这些操作过程中，共获得了42个克隆，其中41个克隆具有预期的产物质粒。结论SfiNX方法适用于对融合和共表达伙伴的不同组合进行反复试验，以优化重组蛋白生产。
ObjectiveConcatenation of twoNdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with anSfiI site was performed to evaluate success rates in construction of polycistronic genes expressible inEscherichia coli.ResultsA series of plasmids with anSfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between theNdeI andXhoI sites of pET-22b were transferred to theSfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.ConclusionThis method, named as theSfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.
翻译起始需要细胞分裂周期 123 (Cdc123) 来促进真核起始因子 2 (eIF2) 的生物合成
DOI：
10.1074/jbc.m113.472290
发表时间：
2013
期刊：
The Journal of Biological Chemistry
影响因子：
--
作者：
Angelika F Perzlmaier;F. Richter;W. Seufert
通讯作者：
W. Seufert
合成生物学：生命设计
影响因子：
64.8
作者：
E. Check
通讯作者：
E. Check
合成生物学：生命设计
影响因子：
64.8
作者：
P. Ball
通讯作者：
P. Ball