title: Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2.
abstract: Glycine transporter GlyT2 contains an extended N-terminal domain which is about three times longer than the N-termini of its closest family members. We previously found that this domain could be separated from the transporter by proteolysis with calpain resulting in the generation of at least two GlyT2N derived peptides. In this work we analyzed the properties of these peptides using bio-informatics, by expressing them in mammalian cell lines and by overexpressing them in bacteria. When expressed in mammalian cell lines, these peptides show widespread localization in the cytoplasm. Their unusually high number of alanine, proline and glycine residues suggests that they posses significant disorder and conformational flexibility, which is supported by their thermal resistivity. Making use of these phenomena, we developed a simple purification method for obtaining pure recombinant GlyT2N derived calpain fragments without using an affinity tag. This procedure can be used to obtain peptide fragments in large amounts for structural, interaction studies or for determining their potential biological activity.
authors.affiliation: Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovakia. Frantisek.Jursky@savba.sk
authors.forename: Frantisek
authors.initials: F
authors.lastname: Jursky
authors.forename: Martina
authors.initials: M
authors.lastname: Baliova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D002460:Cell Line; D019295:Computational Biology; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D050487:Glycine Plasma Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; D010446:Peptide Fragments; D010455:Peptides; D002154:Calpain