Separation of Human Serum Albumin from Recombinant Oryza sativa by Using Aqueous Two-Phase System

• Main Authors: Chih-Chi Wang, 王芝淇
• Other Authors: Chi-Wei Lan
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/39477106488766319525

碩士 === 元智大學 === 化學工程與材料科學學系 === 101 === With the development of genetic engineering technology, proteins for medical application can be expressed in myriads of host. However, the development of purification processes still becomes the major challenge to the applicability of ezpressed protein for biopharmaceutical uses. In fact, the protein purification processes usually employ chromatographic method for product recovery. Howere, the chromatographic technology still has the disadvantages of time-consumping and economically infeasible, leading to problem foe industrial uses. Therefore, to establish the purification process os cost-effectiveness, relatively simple operation and high efficiency in recovery of biomolecules is inevitably of great importance. In this study, the recombinant Oryza sativa L. cv Tainung 67, a provided from Plant Physiology and Culture Laboratory, YZU, was employed as an expression host for human serum albumim (HSA) production. The HSA were produced in the culture medium by the rice suspension cell culture with repeated sucrose provision-depletion cycles. Subsequently, a single purification by using aqueous two-phase systems (ATPS) was conducted to recover the expressed protein from culture feedstock. First, the effects of pH value, binodal curve, tie-line length (TLL), centrifugation speed, ionic strength, operational temperature and volume ratio of ATPS upon recovery performance were considered for system assessment. The results revealed that the optimal conditions for OsrHSA recovery from crude feedstock are by applying ATPS consisted of 18% (w/w) PEG 4000 and 8% (w/w) PO4 at pH 7.5 under room temperature. The efficiency of recovery achieved 69.83 %. The findings illustrated that ATPS could be a promising technique to recover HSA protein in one-step operation. In addition, to the best of our knowledge this study is the first-attempt work in purification of OsrHSA using ATPS technique. Next, to determine the characteristic of HSA partitioning, the response surface methodology (RSM) was also employed to study the separation of HSA in ATPS. The results show that the most significant factor was interactive factor of two-component composites. According to the RSM model of prediction of selectitvly, the reaponse equation Y = 0.9984 - 0.111642 X1 - 0.448368 X2 - 0.123388 X12 + 0.043 X1X2 +0.1503625 X22 was obtained, and the model was confirmed via triplicate experiments afterwards.