A Strategy for Synthesis of Pathogenic Human Immunoglobulin Free Light Chains in E. coli

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.

The Kinetic Stability of a Full-Length Antibody Light Chain Dimer Determines whether Endoproteolysis Can Release Amyloidogenic Variable Domains

“…Full-length LCs were expressed as inclusion bodies in E. coli , as described previously [11, 32]. BL21 (DE3) E. coli were grown in ZYP-5052 autoinduction media [53] containing glucose, lactose and ampicillin for 24 h at 37 °C, 250 rpm.…”

Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis

“…Monoclonal LCs from multiple patients with myeloma (without amyloid deposition or LC‐related organ dysfunction) served as controls. LCs were isolated from 24 h urine collections as described (28, 29) (Supplemental Data). Each LC lot was tested for bacterial endotoxins with limulus amebocyte lysate assay (Pierce).…”

A Caenorhabditis elegans–based assay recognizes immunoglobulin light chains causing heart amyloidosis

“…In addition, mass spectrometry analysis, physicochemical studies, and the examination of the oligomerization state were performed on prototypic proteins, ie, recombinant cardiotoxic (H3-r) and noncardiotoxic (K3-r) amyloidogenic proteins and on BJ proteins purified from urine of a patient suffering from AL cardiomyopathy (H6-BJ) and from a patient with MM (MM2-BJ) (supplemental Methods). 18 The biochemical characteristics of these proteins (eg, size, molecular mass, and calculated isoelectric point) are reported in supplemental Figure 1. All LCs included in the study were l isotype, which represents ;75% of amyloidogenic LCs.…”