Smart DeltaPhase Tags for Purification of Recombinant Peptides

描述（由申请人提供）：鉴于目前长肽（30-70个氨基酸）合成方法的局限性，多肽药物的重要性日益增加，以及对低成本表达和易于纯化多肽的明确需求，本I期研究计划的目标是开发高纯度、高产率、研究规模的功能活性肽的重组表达和非色谱纯化。成本低，与其他化学或生物合成方法生产的肽具有相似的生物活性，但成本明显较低。使Phase Bioscience能够实现这一目标的技术是delta Phase（TM）系统，这是一种新颖的平台技术，用于高水平表达和简单的非色谱纯化，使用一类称为弹性蛋白样多肽（ELPs）的多肽标签。该技术的基本基础是发现融合到ELP的蛋白质和肽保留了ELP的环境触发（温度，盐）可溶-不溶性相变行为，并且这种可逆相变可以在细胞裂解液中进行，通过几个相变循环将ELP融合物与其他细胞蛋白分离到均匀性。该技术的影响将如下：开发新药。由于通过功能基因组学确定了独特的药物靶点，因此需要对它们进行表达和纯化以进行评估（靶点验证）和筛选（高通量筛选和x射线晶体学）。PhaseBio技术将通过以下方式迅速扩大目标肽库：1)首次实现具有成本效益的重组肽表达；2)通过在短短几周内同时生产多达96种肽变体，促进了肽变体的评估；3)消除了由于制造挑战和成本而花费长时间寻找“较短”活性肽的需要；4)实现简单的四步制造工艺的快速规模化。该项目的具体目标包括：1。表达5种与药物相关的重组融合肽；2. 纯化肽至至少90%的纯度，通过LC-MS测定。3. 与市售化学合成或重组肽生产商生产的相同肽相比，通过Phase Bio技术生产的肽具有相似的生物活性。

DESCRIPTION (provided by applicant): Motivated by the limitations of current synthesis methods for long (30-70 amino acids) peptides, the increasing importance of peptide drugs, and the clear need for low-cost expression and easy purification of peptides, the goal of this Phase I research proposal is to develop the recombinant expression and non-chromatographic purification of functionally active peptides at research-scale quantities at high purity levels, high yield, low cost and at similar biological activity to peptides produced by other chemical or biosynthetic methods, but with significantly lower cost. The enabling technology that will allow Phase Bioscience to achieve this objective is the delta Phase(TM) System, a novel platform technology for high-level expression and simple, non-chromatographic purification using a class of polypeptide tags, termed elastin- like polypeptides (ELPs). The fundamental basis of this technology is the discovery that proteins and peptides fused to an ELP retain the environmentally triggered (temperature, salt) soluble-insoluble phase transion behavior of ELPs, and that this reversible phase transition can be carried out in cell lysate to separate ELP fusions from other cellular proteins to homogeneity by a few phase transition cycles. The impact of the technology will be as follows: Development of Novel Medicines. As unique pharmaceutical targets are identified through functional genomics, they will need to be expressed and purified for evaluation (target validation) and screening (high- throughput screening and X-ray crystallography). The PhaseBio technology will rapidly expand the pool of target peptides by 1) enabling first-time, cost-effective expression of recombinant peptides; 2) facilitating the evaluation of peptide variants by enabling the production of up to 96 peptide variants simultaneously in only a matter of weeks; 3) eliminating the need to spend long development time looking for "shorter" active peptides due to manufacturing challenges and costs; and 4) enabling the rapid scale-up of a simple 4-step manufacturing process. The Specific Aims of the project include: 1. Express five pharmaceutically-relevant recombinant fusion peptides; 2. Purify the peptides to at least 90% purity, as measured by LC-MS. 3. Demonstrate similar bioactivity of peptides produced via Phase Bio's technology compared to the same peptides produced by commercially available chemical synthesis or recombinant peptide manufacturers.