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

Protein Expression and Purification
Venkatraman RamanujamSri Rama Koti Ainavarapu

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 isoto...Continue Reading

References

May 9, 2008·Nucleic Acids Research·Christian ColeGeoffrey J Barton
May 15, 2008·BMC Structural Biology·Carolina Perez-Iratxeta, Miguel A Andrade-Navarro

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Citations

Apr 15, 2014·PloS One·Venkatraman RamanujamSri Rama Koti Ainavarapu

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