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.
Highlights
Human immunoglobulin light chains (LC) consist of two domains, each of 100-110 amino acids (12 kDa, approximately), that fold independently of each other: the variable region domain (VL), generated via rearrangement of a V to a J gene segment, and the constant region domain (CL), of minimal variation
To obtain the original full-length monoclonal LC (VL + CL, approximately 650 bp, from codons +1 to +216), standard RT-PCR was employed using the same marrow RNA, a forward patient-specific primer and a universal reverse Cl carboxyterminal cloning primer, corresponding to the last amino acids of CL
In this paper we describe a strategy to obtain significant amounts of human immunoglobulin free LC by the use of a bacterial cell factory
Summary
Human immunoglobulin light chains (LC) consist of two domains, each of 100-110 amino acids (12 kDa, approximately), that fold independently of each other: the variable region domain (VL), generated via rearrangement of a V to a J gene segment, and the constant region domain (CL), of minimal variation. In the present paper we devised a novel, straightforward strategy comprising inclusion body formation as a natural way to obtain consistent amounts of recombinant proteins virtually free of contaminants, with carefully optimized conditions for proper refolding and disulfide bond formation in vitro.
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