Artificial extracellular matrix proteins containing phenylalanine analogues biosynthesized in bacteria using T7 expression system and the PEGylation.
Artificial extracellular matrix proteins containing phenylalanine analogues biosynthesized in bacteria using T7 expression system and the PEGylation.
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DOI：
10.1021/bm200522d
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发表时间：
2011-09
影响因子：
6.2
通讯作者：
A. Takasu;S. Kondo;Akihiro Ito;Y. Furukawa;M. Higuchi;T. Kinoshita;Inchan Kwon
中科院分区：
文献类型：
--
作者：
A. Takasu;S. Kondo;Akihiro Ito;Y. Furukawa;M. Higuchi;T. Kinoshita;Inchan Kwon
使用携带具有扩大的结合口袋的突变型苯丙氨酰-tRNA合成酶（PheRS）的细菌表达宿主，完成苯丙氨酸（Phe）类似物到人工细胞外基质蛋白（aECM-CS5-ELF）中的体内掺入。虽然T5启动子控制下的Ala 294 Gly/Thr 251 Gly突变体PheRS（PheRS**）允许将一些Phe类似物掺入蛋白质中，但T5系统不适合材料科学研究，因为由于T5启动子的中等强度，产生的材料量不足。通过使用一对T7启动子和T7 RNA聚合酶可以克服这一限制。然而，在T7表达系统中，由于Phe类似物与合成活性T7 RNA聚合酶所需的残留Phe竞争掺入，难以实现Phe类似物的高掺入水平。在本研究中，我们制备了T7启动子下的PheRS**，并优化了培养条件，以提高重组aECM蛋白的掺入水平和Phe类似物的掺入水平。在对叠氮苯丙氨酸、对碘苯丙氨酸和对乙酰苯丙氨酸的情况下，掺入和表达水平倾向于增加。我们评估了较低的临界转变温度，这是依赖于掺入比和浊度降低时，掺入水平增加。圆二色性测定表明，这种趋势是基于构象从无规卷曲到β-转角结构的变化。我们证明，聚乙二醇（PEG）可以结合在反应位点的苯丙氨酸类似物纳入。我们还证明了通过PEG缀合制备的aECM-CS5-ELF中弹性蛋白样序列的亲水性增加可以抑制人脐静脉内皮细胞（HUVEC）的非特异性粘附。
In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).