title: Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase.
abstract: The use of Escherichia coli protein expression systems has many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing'Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.
authors.affiliation: Fusion Antibodies Ltd., Springbank Industrial Estate, Belfast BT17 0QL, UK.
authors.forename: Jill
authors.initials: J
authors.lastname: Caswell
authors.forename: Philip
authors.initials: P
authors.lastname: Snoddy
authors.forename: Darragh
authors.initials: D
authors.lastname: McMeel
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Buick
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Scott
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005822:Genetic Vectors / Q000096:biosynthesis; D006801:Humans; D037102:Lectins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D037102:Lectins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C114531:SIGLEC5 protein, human; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases