Type of Document Thesis Author Kasprzak, Agnieszka Joanna URN etd-07282005-152615 Title Increasing the Yield of Thy-1 EGFP Expression in Insect and Mammamlian Cells Degree Master of Science Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Hong Li Committee Member John Dorsey Committee Member Timothy Logan Committee Member Keywords
- Thy-1 Yield
- Baculovirus Expression
- Lec-1 Expression
Date of Defense 2005-07-11 Availability unrestricted AbstractThy-1 is being pursued as a model system for NMR studies of structure and dynamics of intact glycoproteins. However, the low expression yield of the Thy-1 EGFP glycoprotein in insect and mammalian cells has been an obstacle for these studies. The objective of the following studies is to increase the yield of purified Thy-1 EGFP with correct post translational modifications and homogeneity in the three glycosylation sites.
To improve the yield in the baculovirus based insect cell expression systems we investigated the effect of viral titer, multiplicity of infection (MOI), cell density, and length of expression. The MOI and cell density need to be optimized to give the highest yield of recombinant protein. It was found that the titer of a virus needs to be at least 1X108 pfu/ml and that more than two amplifications decrease both titer and expression levels. The optimal MOI used in for virus amplification was found to be between 0.01 to 0.05, while for expression, the optimal MOI is between 1-5. Optimal cell density for amplification of virus is 2.5 X106 cells/ml, while expression cell density is optimal at 3.0 X106 cells/ml. Optimized time of expressing Thy-1 EGFP is 72 hours post infection. Using these optimized conditions, the maximum yield of Thy-1 was 500 ƒÝg per liter of culture.
Mammalian cell expression in Lec-1 cells provides a more homogenous glycosylation pattern on Thy-1 EGFP than observed from wild type CHO cells. In lec-1 cells the factors investigated to optimize the expression yield were media volume, time of expression and cell confluency. The optimal conditions for expression were found to be 48 hours in 50 mls of serum free media with 2mM butyric acid and 20mM HEPES. The cell confluency should be 100% before initiating expression. Using these optimized conditions the maximum protein yield was 2 mg of Thy-1 per liter of expression media.
In a final set of experiments we sought to inhibit the activity of proteases that co-purify with Thy-1 through most of the purification steps. We screened for the effect of protease inhibitors such as E-64, leupeptin, aprotinin, and PMSF to minimize the degradation during purification. Protease activity was analyzed via zymogram assay and PMSF yielded the highest inhibition at a 0.5 mM concentration. The other protease inhibitors were less or completely ineffective.
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