A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli

PLoS One. 2013 Sep 27;8(9):e76022. doi: 10.1371/journal.pone.0076022. eCollection 2013.

Abstract

Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid / biosynthesis
  • Amyloid / ultrastructure
  • Amyloidosis / genetics
  • Amyloidosis / metabolism*
  • Bence Jones Protein / genetics
  • Chromatography, Gel
  • Circular Dichroism
  • Cloning, Molecular
  • DNA Primers / genetics
  • Escherichia coli / metabolism*
  • Genetic Vectors / genetics
  • Humans
  • Immunoglobulin Light Chains / biosynthesis*
  • Immunoglobulin Light Chains / genetics
  • Inclusion Bodies / metabolism
  • Microscopy, Electron
  • Protein Engineering / methods*
  • Protein Folding
  • Recombinant Proteins / biosynthesis*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Amyloid
  • DNA Primers
  • Immunoglobulin Light Chains
  • Recombinant Proteins
  • Bence Jones Protein

Grants and funding

This work was supported by Fondazione IRCCS Policlinico San Matteo, Grant 2009-2012 by AIRC (Associazione Italiana per la Ricerca sul Cancro) Special Program Molecular Clinical Oncology 5 per mille n. 9965, Italian Ministry of Health (GR-2010-2317596), Amyloid Foundation and Fondazione Mintas, Ghislieri College, Pavia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.