title: Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans.
abstract: βγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two β-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens βγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens βγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
authors.forename: Venkatraman
authors.initials: V
authors.lastname: Ramanujam
authors.forename: Kandala V R
authors.initials: KV
authors.lastname: Chary
authors.forename: Sri Rama Koti
authors.initials: SR
authors.lastname: Ainavarapu
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D017020:Methanosarcina / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013057:Spectrum Analysis; D038204:beta-Crystallins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D002135:Calcium-Binding Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D038204:beta-Crystallins