Peptide Synthesis and Purification Core Facility

仪器仪表。该设施拥有三种蛋白质技术自动多肽 一台先进的化学合成器和一台先进的ACT90多肽合成器。而蛋白质 技术多肽合成器由于在氨基酸树脂上加入10倍的过量氨基酸，可以在较短的时间内提供少量的多肽(以镁为单位)，ACT可以提供10g多肽，但过程很慢，因为在合成的每个阶段反应完成后，每天添加一个氨基酸。 由于ACT 90中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。对于多肽的提纯，核心设施拥有三个Beckman分析型高效液相色谱法和两个制备型高效液相色谱仪(Beckman和Varian)。除此之外，核心设施使用BioRad FPLC系统，这些系统连接到Michel Muller中压液相色谱(MPLC)玻璃柱。这些色谱柱的大小各不相同，最小的(分析)包含12g的固定相(C-4或C-18硅胶)，最大的柱包含800克的“自制”反相固定相(C-4和C-18反相硅胶)。我们通常用不同链长的脂肪酸来烷化硅胶。我们一次生产2公斤到4公斤的自制柱用固定相。这提供了更高的能力来净化大型 合成多肽的数量。这些柱子也可用于纯化重组蛋白。一个典型的过程将在方法一节中描述。

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.