TY - JOUR
T1 - Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains
AU - Misra, Pinaki
AU - Tischer, Alexander
AU - Lampe, Lindsey
AU - Pierluissi-Ruiz, Valeria
AU - Dick, Christopher J.
AU - Bragantini, Benoit
AU - Kormshchikov, Nikita
AU - Auton, Matthew
AU - Ramirez-Alvarado, Marina
N1 - Publisher Copyright:
© 2024 Elsevier B.V.
PY - 2024/5/1
Y1 - 2024/5/1
N2 - Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.
AB - Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.
KW - AL amyloidosis
KW - AL full-length proteins
KW - Circular dichroism
KW - Fluorescence
KW - Mammalian cell protein expression
UR - http://www.scopus.com/inward/record.url?scp=85181823400&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85181823400&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2023.140993
DO - 10.1016/j.bbapap.2023.140993
M3 - Article
C2 - 38169170
AN - SCOPUS:85181823400
SN - 1570-9639
VL - 1872
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 3
M1 - 140993
ER -