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Expression of recombinant canine sFlt1 as an experimental anti‐angiogenic agent
Author(s) -
Payne Laura B.,
Herring Ian P.,
Huckle William R.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.682.12
Subject(s) - recombinant dna , microbiology and biotechnology , sf9 , glycosylation , biology , transfection , chemistry , gene , biochemistry , spodoptera
Formation of new blood vessels is regulated by pro‐ and anti‐angiogenic factors, including VEGF and its secreted receptor sFlt1. With the goal of evaluating sFlt1 as a therapeutic for ocular diseases in dogs, we undertook the generation of recombinant canine sFlt1 (rcsFlt1) protein. Canine sFlt1 coding sequence was recovered as recombinant baculovirus after transfection into Sf9 cells. rcsFlt1 proteins were purified from infected Sf9 conditioned media by adsorption to heparin‐agarose and resolved by SDS‐PAGE. Immunoblotting of heparin‐purified conditioned medium revealed rcsFlt1 of predicted molecular weight (~115 kDa). rcsFlt1 migrated on SDS‐PAGE as a species apparently larger than recombinant mouse sFlt1 similarly produced. Endoglycosidase treatment of canine and mouse rsFlt1 reduced both to co‐migrating species of ~80 kDa, consistent with their predicted primary structures. This result, together with the presence of more predicted N‐glycosylation sites on canine than mouse sFlt1, suggests that differential glycosylation accounts for differential migration of canine and mouse forms. Future studies will examine the topical tolerability of purified rcsFlt1 on canine eyes. Supported by VMRCVM RGS