Title page for ETD etd-06302008-195006

Type of Document Master's Thesis
Author Ross, Kristin Coby
URN etd-06302008-195006
Title Separation of Recombinant β-Glucuronidase from Transgenic Tobacco by Aqueous Two-Phase Extraction
Degree Master of Engineering
Department Biological Systems Engineering
Advisory Committee
Advisor Name Title
Zhang, Chenming Mike Committee Chair
Agblevor, Foster Aryi Committee Member
Goldstein, Aaron S. Committee Member
  • transgenic tobacco
  • recombinant β-glucuronidase
  • aqueous two-phase extraction
  • protein purification
Date of Defense 2008-05-19
Availability restricted
Separation of Recombinant ß-Glucuronidase from Transgenic Tobacco by Aqueous Two-Phase Extraction

Kristin Coby Ross


Biopharmaceutical manufacturing is a rigorous and expensive process. Due to the medicinal nature of the product, a high purity level is required and several expensive purification steps must be utilized. Cost-effective production and purification is essential for any biopharmaceutical product to be successful and development of the fastest, most economical, and highest-yielding purification scheme is a constant engineering challenge. Commercial-scale purification schemes currently revolve around the use of multiple chromatography steps for the purification of biopharmaceutical products. Chromatography has many shortcomings including high cost, limited throughput, and complex scale up. The goal of this research was to develop an alternative, non-chromatography purification step for the separation of an acidic model protein, recombinant ß-glucuronidase (rGUS), from transgenic tobacco with high yield and purity.

Aqueous two-phase extraction (ATPE) is a powerful technique for separation and purification of proteins, and has the potential to replace an expensive chromatography step for the initial purification of recombinant proteins. ATPE enables high levels of target protein recovery and concentration while removing large amounts of impurities from the initial extract. Fractional factorial designs and response surface methodology were used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4 % (w/w) PEG/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process.

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