Production and purification of self-assembling peptides in Ralstonia eutropha
Production and purification of self-assembling peptides in Ralstonia eutropha
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DOI：
10.1016/j.pep.2005.08.023
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发表时间：
2006-04-01
影响因子：
1.6
通讯作者：
Gerngross, TU
中科院分区：
文献类型：
--
作者：
Reed, DC;Barnard, GC;Gerngross, TU
自组装肽已成为一种有吸引力的组织工程支架材料，但与固相化学合成相关的费用限制了它们的广泛应用。此外，化学合成的保真度限制了可以通过该方法均匀产生的多肽的长度。通过重组DNA技术的模板衍生的生物合成可以克服这两个问题。然而，从重组蛋白表达系统回收多肽通常涉及多步纯化方案。在这项研究中，我们报告了一个整合的方法来重组生产和纯化自组装肽从最近开发的表达宿主真养罗尔斯通氏菌。纯化是基于碳水化合物结合模块（CBM）对纤维素的特异性亲和力。在第一步中，我们通过组装绿色荧光蛋白（GFP）和CBM的融合文库并测定无细胞提取物的荧光来鉴定在真养反式酵母中表达良好的CBM。发现三种GFP：：CBM融合物以与单独GFP相似的水平表达，其中两种CBM能够介导GFP：：CBM融合物的纤维素结合。然后将这两个CBM融合到自组装肽RAD 16-I：：E（（N）-RADARADARADAE-（C））的多个重复序列上。融合蛋白CBM：：E：：（RAD 16-I：：E）（4）在R.真养型，并使用CBM对纤维素的亲和性进行纯化。随后用内切蛋白酶GluC进行蛋白水解裂解，释放出具有与化学合成的对应物RAD 16-I相似性质的RAD 16-I：：E肽单体。(c)2005年爱思唯尔公司All rights reserved.
Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E ((N)-RADARADARADARADAE-(C)). The fusion protein CBM::E::(RAD16-I::E)(4) was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I. (c) 2005 Elsevier Inc. All rights reserved.