毕赤酵母系统来源的重组人血清白蛋白的制备及初步晶体学分析

Preparation and Crystallographic Studies of Recombinant Human Serum Albumin in Pichia pastoris

人血清白蛋白(human serum albumin,HSA),为血浆中含量最丰富的蛋白质,具有调控血液中的功能蛋白质、脂肪酸、激素、药物和维持血液渗透压功能。毕赤酵母工程菌Pichia pastoris GSll5/p PIC9K-rHSA经50L发酵罐发酵,发酵液冷冻离心,上清液超滤浓缩后,依次通过Blue Sepharose 6FF亲和层析、Phenyl Sepharose 6FF疏水层析和SP Sepharose 6FF离子层析纯化,实现rHSA的规模化制备,最终rHSA的纯度可达99.9%以上。通过rHSA的结晶条件筛选实验,筛选出沉淀剂分别为PEG 1500、PEG 3350、PEG 6000、MPEG 2000和MPEG 5000五种结晶条件,结晶环境均为疏水性环境,并且结晶环境不能含有类似DTT的强还原剂。其中沉淀剂为MPEG2000条件下优化出的rHSA和pHSA蛋白晶体经X光衍射分辨率最好,分别为3.4魡和3.1魡,通过分子置换法得到rHSA和pHSA的晶体结构,二者结构大体相似,为rHSA在临床上的应用和HSA融合蛋白的晶体结构研究提供可靠的依据和基础。

Human serum albumin（HSA）,the most abundant protein in plasma,with functions of regulating blood protein,fatty acids,hormones,drugs,and representing the main determinant of plasma oncotic pressure,providing a depot and carrier for many endogenous and exogenous compounds.The engineering pichia pastorios GSll5/p PIC9K-rHSA was fermented by 50L fermentor.Highly purified rHSA was separated from fermentation supernatant by ultra filter,Blue sepharose affinity chromatography,Phenyl sepharose hydrophobic chromatography and SP sepharose ion exchange chromatography purification.Eventually the purity of rHSA can reach more than99.9%.Through screening crystallization conditions of rHSA,five conditions with precipitant of PEG1500,PEG 3350,PEG 6000,MPEG 2000 and MPEG 5000 were obtained.The crystallization condition was hydrophobic and without strong reductant like DTT.Crystals of rHSA and pHSA with precipitant of MPEG 2000 were best for X ray diffraction,respectively the resolution were 3.4A and 3.1A.Crystal structure of rHSA and pHSA were the same as a whole that obtained through molecular displacement method.The results provided reliable basis and foundation for the clinical application of rHSA and crystal structure study ofHSA fusion proteins.