Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins
Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins
复制标题
DOI：
10.1128/aem.02595-14
复制
发表时间：
2015-01-01
影响因子：
4.4
通讯作者：
Rehm, Bernd H. A.
中科院分区：
文献类型：
--
作者：
Hay, Iain D.;Du, Jinping;Rehm, Bernd H. A.
提供了显示各种可定制亲和蛋白结合域的聚酯树脂的体内细菌生产的概念证明。这是通过在细菌聚酯合成酶中设计各种蛋白质结合域来实现的。基于不同结构折叠和来自分子文库的亲和结合域被用来证明该技术的潜力。设计锚蛋白重复序列蛋白（DARPins），工程OB-fold结构域（OBodies）和V-HH结构域来自骆驼抗体（纳米体）。在单个细菌发酵步骤中生产了相应的树脂，并制定了简单的纯化方案。纯化后的树脂适用于大多数实验室规模的亲和层析。所有的亲和结构域测试产生的聚酯珠具有特定的亲和力的目标蛋白。这些亲和树脂的结合能力范围从每湿克聚酯亲和树脂90到600 nmol的蛋白质，使得从复杂的细菌细胞裂解物中纯化重组蛋白的纯度在一步中达到96%。通过传统的实验室规模摇瓶发酵有效地生产聚酯树脂，导致细菌积累高达其细胞干重的55%为聚酯。通过细胞内特定亲和树脂及其靶标的协同生产，进一步证明了该技术的实用性。这使得在体内结合和纯化共同产生的“目标蛋白”成为可能。总的来说，这项研究为利用分子工程的聚酯合成酶来实现先前选择的结合域的特定生物分离树脂的微生物生产提供了证据。
Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and V-HH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.