Rapid assembly of synthetic genes encoding protein polymers
Rapid assembly of synthetic genes encoding protein polymers
复制标题
DOI：
10.1021/ma981660f
复制
发表时间：
1999-06-01
期刊：
影响因子：
5.5
通讯作者：
Conticello, VP
中科院分区：
文献类型：
--
作者：
McMillan, RA;Lee, TAT;Conticello, VP
描述了一种基于无缝克隆技术的合成编码蛋白质聚合物基因的快速组装的通用方法。使用该技术构建了编码弹性蛋白模拟多肽[(Val-Pro-Gly-Val-Gly)(4)(Val-Pro-Gly-Xaa-Gly)](XaA=Lys 1；Ile，2)的重复序列的基因。这种方法的使用消除了连接异构化对有限的非回文限制性内切酶池的依赖，并减少了亚克隆步骤的数量。克隆了一个全长约3000个碱基对的合成基因，该基因编码一个基于重复序列1的蛋白质聚合体。该基因在重组大肠杆菌中的诱导表达获得了90 kDa的蛋白质聚合体1的高产(mg/L细菌培养)。用固定化金属亲和层析法纯化蛋白，纯化后的蛋白纯度较高。通过N-末端氨基酸序列分析和定点蛋白水解酶切割片段的MALDI-TOF质谱分析，证实了该蛋白质聚合物的序列。
A general method is described for the rapid assembly of synthetic genes encoding protein polymers based on the Seamless cloning technique. Genes encoding repeats of the elastin-mimetic polypeptides [(Val-Pro-Gly-Val-Gly)(4)(Val-Pro-Gly-Xaa-Gly)] (Xaa = Lys 1; Ile, 2) were constructed using this technique. The use of this method eliminates the dependence of the concatamerization on a limited pool of nonpalindromic restriction endonucleases and reduces the number of subcloning steps. A synthetic gene of approximately 3000 base pairs in length was isolated that encoded a protein polymer based on repeat sequence 1. An inducible expression of this gene in bacterial cultures of recombinant E. coli afforded a 90 kDa protein polymer of 1 in high yield (64 mg/L of bacterial culture). The protein was purified to homogeneity using immobilized metal affinity chromatography. The sequence of the protein polymer was confirmed by N-terminal amino acid sequence analysis and MALDI-TOF mass spectrometry of site-specific proteolytic cleavage fragments.