Smart DeltaPhase Tags for Purification of Recombinant Peptides

描述（申请人提供）：由于目前合成方法的局限性，（30-70个氨基酸）肽、肽药物的日益重要性以及对肽的低成本表达和容易纯化的明确需求，该I期研究计划的目标是在研究中开发功能活性肽的重组表达和非色谱纯化，以高纯度水平、高产率、低成本和与通过其它化学或生物合成方法生产的肽类似的生物活性，但成本显著降低。使Phase Bioscience能够实现这一目标的技术是delta Phase（TM）系统，这是一种使用一类多肽标签（称为弹性蛋白样多肽（ELP））进行高水平表达和简单的非色谱纯化的新型平台技术。该技术的基本基础是发现与ELP融合的蛋白质和肽保留了ELP的环境触发（温度、盐）可溶-不溶相转变行为，并且该可逆相转变可以在细胞裂解物中进行，以通过几个相转变循环将ELP融合物与其他细胞蛋白质分离成同质性。该技术的影响将如下：开发新药物。由于通过功能基因组学鉴定独特的药物靶标，因此需要表达和纯化它们以进行评估（靶标验证）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快速扩大靶肽库：1）实现重组肽的首次、具有成本效益的表达; 2）通过在几周内同时生产多达96种肽变体，促进肽变体的评估; 3）消除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;以及4）能够快速放大简单的4步制造工艺。该项目的具体目标包括：1。表达5个与药物相关的重组融合肽; 2.将肽纯化至至少90%纯度，如通过LC-MS所测量。证明通过Phase Bio技术生产的肽与市售化学合成或重组肽生产商生产的相同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.