Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: Biosynthesis and characterization
Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: Biosynthesis and characterization
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DOI：
10.1021/mp049906s
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发表时间：
2005-03-01
影响因子：
4.9
通讯作者：
Ghandehari, Hamidreza
中科院分区：
文献类型：
--
作者：
Haider, Mohamed;Leung, Vivian;Ghandehari, Hamidreza
基因工程技术用于设计和开发可控基因传递的聚合物的独特优势在于对聚合物结构的精细控制。在本文中，我们报道了一系列新的丝状弹性蛋白聚合物(SELP415K)的生物合成和表征，即SELP415K，其每单体单位的弹性蛋白块比先前研究的用于基质介导的基因传递的SELP47K更大。采用了一种新的克隆策略，利用适当的限制性内切酶识别位点，将8个弹性蛋白单元(8E)整合到现有的SELP47K单体基因的DNA序列中。随机多聚体后，选择所需大小的多聚体基因片段，在Ni-琼脂糖柱上表达和纯化。用MALDI-TOF和氨基酸分析分别测定了纯化的SELP的相对分子质量和序列组成。考察了结构变化对聚合物流变性能的影响。此外，从47K和415K-8mer聚合物溶液中制备了水凝胶圆盘，研究了固化时间和环境条件对水凝胶平衡溶胀比随聚合物组成的变化的影响。DNA测序和琼脂糖凝胶电泳法证实成功克隆了SELP415K的312个碱基的单体基因片段。SELP415K单体基因片段的随机串联产生了分别为6、8和10个重复的SELP415K多聚体序列的文库，每个重复序列产生具有准确分子量和序列的聚合物。流变学测试表明，聚合物组成对复合剪切模数(G*)和凝胶点均有影响。对47K和415K-8mer聚合物溶液制备的水凝胶圆盘的平衡溶胀研究表明，聚合物组成的变化导致了不同的凝胶模式，并增加了对温度和离子强度变化的敏感性，但对pH变化没有影响。总之，这些结果证明了重组技术在具有特定结构的工程聚合物中的潜力，这使得研究影响基质介导的基因和生物活性物质传递的结构参数成为可能。
The unique advantage of genetic engineering techniques for the design and development of polymers for controlled gene delivery lies in exquisite control over polymer structure. In this article we report the biosynthesis and characterization of a series of new silk-elastinlike protein polymers (SELPs), namely, SELP415K, with larger elastin blocks per monomer unit than SELP47K previously studied for matrix-mediated gene delivery. A new cloning strategy was used, where a block of eight elastin units (8E) was integrated into the existing DNA sequence of SELP47K monomer genes using appropriate restriction endonuclease recognition sites. Following random multimerization, multimer gene segments of desired size were selected, expressed, and purified on Ni-agarose columns. The molecular weight and sequence composition of the purified SELPs were determined by MALDI-TOF and amino acid analysis, respectively. The influence of structural changes on the rheological properties of the polymers was investigated. In addition, hydrogel disks were prepared from 47K and 415K-8mer polymer solutions, and the effects of cure time and environmental conditions on the hydrogel equilibrium swelling ratio as a function of polymer composition were studied. DNA sequencing and agarose gel electrophoresis confirmed the successful cloning of the monomer gene segment of SELP415K consisting of 312 bp. Random concatemerization of SELP415K monomer gene segments resulted in a library of SELP415K multimer sequences of 6, 8, and 10 repeats respectively, each yielding a polymer with exact molecular weight and sequence. Rheometric measurements showed that both complex shear modulus (G*) and gelation point were influenced by polymer composition. Equilibrium swelling studies on hydrogel disks prepared from 47K and 415K-8mer polymer solutions showed that changes in polymer composition resulted in different gelation patterns and increased sensitivity toward changes in temperature and ionic strength but not pH. Together these results demonstrate the potential of recombinant techniques in engineering polymers with defined structures which allows the study of the structural parameters affecting matrix-mediated delivery of genes and bioactive agents.