Novel vectors for biochemical and functional analyses of secreted
proteins.
Mingliang Zhang (1), Scott E. Erdman (2)
(1) Department of Biology, Syracuse University, 130 College Place,
Syracuse , NY 13244-1220, USA; (2) Department of Biology, Syracuse
University
Secreted proteins frequently contain posttranslational
modifications in the form of N- and O-linked glycan groups, GPI anchor
structures and intrapolypeptide disulfide bridges. Analyses of factors
involved in the biogenesis of these structures and their biochemical
nature, including potentially complicated glycans, are greatly
facilitated by expression systems that allow over expression and permit
purification via affinity tags. To help address this need we have
modified a vector/expression system from Invitrogen, pYD1, which
secretes and 'displays' proteins as fusions to the Aga2 protein which
are disulfide bound to the yeast cell wall resident protein Aga1p.
Depending on the cloning construction, fusions may contain COOH-terminal
Express and V5 epitopes, and/or a 6xHis affinity tag. We have created
vectors containing an alternative LEU2 selection marker that should
allow expression in strains of the set of ORF knock-outs for the genome
(Research Genetics). We further modified one vector by removing Aga2p
sequences other than the signal peptide and cleavage site. This vector
has been successfully used to secrete proteins of interest into the
medium. Such proteins can subsequently be purified to near homogeneity
by two simple steps; MW cutoff membrane concentration and Ni+ affinity
chromatography. We have applied the use of these vectors to study
posttranslational modifications to a conserved domain found in a variety
of fungal adhesins.
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