Peptide Synthesis and Purification Core Facility

肽合成与纯化核心设施

• 批准号: 8242749
• 负责人: G M ANANTHARAMAIAH
• 金额: $ 23.96万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242749
• 关键词: Amino Acids; Atherosclerosis; Core Facility; Fatty Acids; Glass; High Pressure Liquid Chromatography; Home environment; Length; Lipoproteins; Liquid Chromatography; Methods; Peptide Synthesis; Peptides; Phase; Plant Resins; Procedures; Process; Proteins; Reaction; Recombinant Proteins; Staging; System; Technology; Time; cost; fast protein liquid chromatography; instrumentation; operation; pressure; programs; silochrome; synthetic peptide

Instrumentation. The facility possesses three Protein Technology automatic peptide synthesizers and one Advanced ChemTech (ACT 90) peptide synthesizer. While the Protein Technology peptide synthesizers can provide small amounts of the peptides (in mg quantities) in a shorter duration due to the addition of ten times excess of incoming amino acids on to the amino acid-resin, the ACT can provide 10g quantities of peptides but the process is slow since one amino acid is added per day after the completion of reaction is ascertained at each stage of the synthesis. Since the starting resin used in ACT 90 is more, we avoid adding more than the required excess of amino acid derivatives at each stage in order to save costs on amino acid derivatives. For the purification of peptides the core facility possesses three Beckman analytical HPLC and two preparative HPLC units (Beckman and Varian). In addition to this, the core facility utilizes BioRad FPLC systems that are connected to Michel Muller Medium Pressure Liquid Chromatography (MPLC) glass columns. These columns are of various sizes with the smallest (analytical) containing 12g of the stationary phase (C-4 or C-18 silica gel) and the biggest column containing 800g of "home made" reverse phase stationary phase (C-4 and C-18 Reversed Phase silica gels). We routinely alkylate Silica Gel with various chain-lengths of fatty acids. At a time we produce 2 kg to 4kg of the stationary phase for home made columns. This provides a higher capacity to purify large quantities of synthetic peptides. These columns are also used to purify recombinant proteins. A typical procedure will be described in the Methods Section.

仪器仪表。该设施拥有三台Protein Technology自动肽 合成器和一台 Advanced ChemTech (ACT 90) 肽合成器。虽然蛋白质 技术肽合成仪可以在更短的时间内提供少量的肽（以毫克为单位），因为在氨基酸树脂上添加了十倍过量的输入氨基酸，ACT 可以提供 10g 量的肽，但该过程由于在合成的每个阶段确定反应完成后每天添加一个氨基酸，因此速度很慢。 由于ACT 90使用的起始树脂较多，我们避免在每个阶段添加超过所需过量的氨基酸衍生物，以节省氨基酸衍生物的成本。对于肽的纯化，核心设施拥有三台贝克曼分析型高效液相色谱仪和两个制备型高效液相色谱仪（贝克曼和瓦里安）。除此之外，核心设施还利用与 Michel Muller 中压液相色谱 (MPLC) 玻璃柱相连的 BioRad FPLC 系统。这些色谱柱有各种尺寸，最小的（分析用）含有 12 克固定相（C-4 或 C-18 硅胶），最大的色谱柱含有 800 克“自制”反相固定相（C-4 和 C -18 反相硅胶）。我们通常用各种链长的脂肪酸对硅胶进行烷基化。我们一次为自制色谱柱生产 2 公斤至 4 公斤固定相。这提供了更高的净化能力 合成肽的数量。这些柱也用于纯化重组蛋白。典型的程序将在方法部分进行描述。