Smart DeltaPhase Tags for Purification of Recombinant Peptides

用于纯化重组肽的智能 DeltaPhase 标签

• 批准号: 7111935
• 负责人: SUSAN F DAGHER
• 金额: $ 10万
• 依托单位: PHASEBIO PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2006-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111935
• 关键词: cell line; elastin; liquid chromatography mass spectrometry; molecular cloning; protein purification; recombinant proteins; technology /technique development

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.

描述（由申请人提供）：由于目前长肽（30-70个氨基酸）合成方法的局限性、肽药物的重要性日益增加以及对肽的低成本表达和易于纯化的明确需求，这一阶段的研究计划的目标是开发重组表达和非色谱纯化功能活性肽在研究规模的数量，高纯度水平，高产率，低 成本和生物活性与其他化学或生物合成方法生产的肽相似，但成本显着降低。使 Phase Bioscience 能够实现这一目标的技术是 delta Phase(TM) 系统，这是一种新颖的平台技术，使用一类多肽标签（称为类弹性蛋白多肽 (ELP)）进行高水平表达和简单的非色谱纯化。该技术的基本原理是发现融合到 ELP 上的蛋白质和肽保留了 ELP 环境触发的（温度、盐）可溶-不溶相变行为，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质分离，达到同质性。该技术的影响如下： 新药的开发。由于通过功能基因组学鉴定了独特的药物靶标，因此需要对其进行表达和纯化以进行评估（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目标肽库：1) 实现重组肽的首次、经济有效的表达； 2) 通过在短短几周内同时生产多达 96 个肽变体，促进肽变体的评估； 3) 无需因制造挑战和成本而花费较长的开发时间来寻找“较短”的活性肽； 4) 能够快速扩大简单的 4 步制造工艺的规模。该项目的具体目标包括： 1. 表达5种药学相关的重组融合肽； 2. 根据 LC-MS 测定，将肽纯化至至少 90% 纯度。 3. 证明通过 Phase Bio 技术生产的肽与商业化学合成或重组肽制造商生产的相同肽具有相似的生物活性。