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Expression and auto‐processing of hedgehog‐like proteins from Brugia malayi
Author(s) -
Savidis George,
Drago Matthew J.,
Reitter Julie N.,
Mills Kenneth V.
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.753.2
Subject(s) - intein , hedgehog , biology , fusion protein , computational biology , proteome , microbiology and biotechnology , genetics , signal transduction , rna splicing , gene , recombinant dna , rna
Hedgehog proteins have an N‐terminal signaling domain and a C‐terminal auto‐processing domain, called the HINT domain (for Hedgehog‐INTein, as it shares sequence and structural similarities to self‐splicing inteins). The HINT domain has a C‐terminal sterol‐recognition region (SRR) not shared with inteins. We will analyze the post‐translational cleavage between the signaling and HINT domains from a hedgehog‐like protein from the parasitic nematode Brugia malayi . We have created E. coli expression vectors that encode fusion proteins of 1) an N‐terminal maltose‐binding protein to the HINT domain, or 2) an N‐terminal His‐tag to the signaling and HINT domains. We have created both fusion proteins with and without the SRR. We are currently optimizing expression and purification conditions for these proteins, with the aim to study the role of conserved intein residues in the hedgehog‐processing mechanism. This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and a Sherman Fairchild Foundation Summer Research Scholarship (GS).