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Post-translational modifications and expression stability of gpi-anchored and secreted forms of a recombinant metalloproteinase

University of British Columbia

The majority of recombinant proteins are produced as soluble secreted proteins. An alternative approach involves expressing recombinant proteins on the cell surface as glycosylphosphatidylinositol (GPI)-anchored proteins and harvesting with phosphatidylinositolphospholipase C. Leishmania GP63 is a GPI-anchored zinc metalloproteinase that is synthesized as a preproprotein and has three potential sites for N-linked glycosylation. Recombinant GP63 was produced in Chinese hamster ovary (CHO) cells as both a GPI-anchored and secreted protein, to investigate the differences in expression levels, stability and post-translational modifications. The expression of both proteolyrically active GP63 (GP63WT) and an active site mutant of GP63 (GP63E265D) was investigated. Flow cytometry was used to follow the stability of GP63WT and GP63E265D expression on the cell surface. Expression of GP63WT was found to be unstable whether or not methotrexate (MTX) selection was maintained. In contrast expression of GP63E265D was stable under MTX selection. In the absence of selection the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Loss of GP63 expression resulted in higher growth rate nonproducer populations. Expression of recombinant GP63 on the surface of CHO cells enabled a quantitative approach to evaluate expression stability during long-term culture. Comparison of producing and nonproducing populations has given insight into the mechanisms involved in loss of recombinant protein expression. Secreted GP63 production rates were over 20 fold higher than membrane expressed protein, however the post-translational modifications of secreted and membrane expressed proteins were different. GP63 was secreted in a latent form that retained the pro-region normally processed during cell surface expression. Fast atom bombardment-mass spectrometry of the N-linked glycans isolated from both secreted and membrane expressed GP63 showed that the predominant structures were complex biantennary types, however the glycan profiles showed dramatic differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained poly-N-acetyllactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. These changes in post-translational modifications represent important factors to be considered when modifying membrane expressed protein for secreted production.

Thesis/Dissertation

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2009-04-20

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http://hdl.handle.net/2429/7443

Microbiology and Immunology

University of British Columbia

UBCV

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