Design And Resolution Of A Simple-Type Column For Renaturation With Simultaneous Purification Of Proteins And Its Application

A new kind of chromatographic column called simple-type chromatographic column was designed and investigated by using the packings of hydrophobic interaction chromatography with large particles in this thesis. Lysozyme was selected as a model protein for investigating the renaturation, recoveries of mass and bioactivity and found out to be 96.6+1.3% and 101.1+6.0%, respectively. In the same time, the simple-type chromatographic column was used the renaturation with simultaneous purification of recombinant human interferon-r,Cytocrirome C in pig-heart and rude a-Amylase, and all these have obtained satisfied results. This thesis includes four parts as follows:1. Design and Resolution of the Simple-Type Column for Renaturation with Simultaneous Purification of Proteins and Its Application to Lysozyme RefoldingBased on short-column theory of biopolymers and stoichiometric displacement theory for retention (SDT-R) of solute, a new kind of simple-type column for renaturation with simultaneous purification of proteins was designed, called simple-type chromatographic column. The cost of the column is decreased significantly by using plastic as column tube and using the bonded silica with large particle as stationary phase. Moreover, the column can work well under low pressure, easily operation and even without chromatograph. The simple-type column of hydrophobic interaction chromatography (HIC) was proved to have a good resolution, renaturation and high recoveries of mass and bionactivity by separating standard proteins and the renatured lysozyme(Lys).2. Renaturation with Simultaneous Purification of rhIFN-γThe simple-type column for renaturation with simultaneous purification of proteins was used in the renaturation and purifiacation of rhIFN-y, the chromatographic condition employed was optimized. A good result was obtained by nonlinear gradient mode. The purify of rhIFN-γ was 94% and specify activity was 3.2x108IU mg-1 in 50min with only one step purification.3. Purification of Cyt-C from pig-heartThe simple-type HIC column was used in Cyt-C purifiacation of pig-heart. Without a series of pre-course step by traditional method, the purify of purified Cyt-C by the presented method is 94% and mass recovery is 96.2% with also only one step purification. The whole process is simple and fast, moreover, the mass recovery is very high.4. Purification of a -AmyIn this study, rude a-Amylase was purified by the simple-type HIC column. The character of the presented method is easily operation, fast separation and high efficiency of purification. But the bioactivity recovery is not high, and how to raise the recovery of bioactivity of a -Amy should be investigated further.