Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography–multiangle laser light scattering

Ye, Hongping

doi:10.1016/j.ab.2006.05.025

Cited by 92 publications

(51 citation statements)

References 28 publications

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“…However, insoluble aggregates are not considered to be measurable by SEC due to potential removal via filtration by the column or precolumn or by the sample preparation for SEC (e.g., centrifugation). Factors such as protein shape, protein glycosylation or pegylation 90 could affect the accuracy if the molecular weight of protein species is determined based on a calibration curve using calibration standards. 91 Well characterized, water-soluble and globular proteins are used as calibration standards, which may differ in their elution properties in comparison with the protein of interest.…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

Protein aggregation: Pathways, induction factors and analysis

Mahler

Frieß

Grauschopf

et al. 2009

Journal of Pharmaceutical Sciences

748

635

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Section: Size Exclusion Chromatographymentioning

confidence: 99%

Protein aggregation: Pathways, induction factors and analysis

Mahler

Frieß

Grauschopf

et al. 2009

Journal of Pharmaceutical Sciences

748

635

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“…Since the amount of light scattered is directly proportional to the product of the weight-average molar mass and the macromolecule concentration, SEC coupled with multi-angle laser light scattering (SEC-MALS) has been used to characterize the molecular mass of individual species, size distribution within the peaks, and extent of aggregation and degradation as evidenced by molecular weight increase or reduction and the change in monomer amount. 8,9 Sodium dodecyl sulfate PAGE (SDS PAGE) is a traditional technique used to separate different size proteins according to their electrophoretic mobility differences. 10 In 1999, a novel platform based on microfluidics technology with LabChip, the Agilent 2100 Bioanalyzer, was introduced to convert qualitative SDS-PAGE to quantitative electropherogram.…”

Section: Introductionmentioning

confidence: 99%

Characterization of monoclonal antibody size variants containing extra light chains

Lü

Liu

et al. 2013

mAbs

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“…S3C) was overexpressed in Escherichia coli, and the purified proteins were further investigated by a range of biophysical techniques. Size-exclusion chromatography coupled to multiangle laser light scattering (35) revealed the stoichiometry of both receptor-HEL complexes to be consistent with two Ig domains bound to a single HEL molecule (Fig. S3 A and B).…”

Section: Structural Reconstruction and Selection Of Reconstructed Primentioning

confidence: 99%

Structural reconstruction of protein ancestry

Rouet

Langley

Schofield

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B-and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence. protein evolution | protein structure | directed evolution | antibody | homodimer T he adaptive immune system of jawed vertebrates provides specific responses to pathogens and forms long-lasting immunological memory of past encounters (1). Key components of this system are B and T lymphocytes and their cognate receptors: B-cell receptor (membrane-bound or secreted as antibodies) and T-cell receptor. Both receptors are heterodimeric molecules formed by the association of Ig domain building blocks, which then assemble into higher order complexes. This behavior is exemplified by the canonical Y-shaped IgG antibody molecule, composed of two heavy chains (each containing four Ig domains) and two light chains (each containing two Ig domains) (2). Within the N-terminal Ig domains of the receptors, hypervariable complementarity determining regions (CDRs) mediate contact with antigen (Fig. 1A). The diversity and specificity of these receptors is based on the genetic rearrangement of variable (V), diverse (D) and joining (J) gene segments, mediated by recombination-activating gene enzymes, with further diversity introduced through somatic hypermutation (3).Although the molecular mechanisms and structural features of rearranging Ig antigen receptors are well documented, only limited insights have been gained into their evolutionary origins (4, 5). Reconstructing the evolutionary history of antigen receptors using molecular phylogenetic analysis has proven difficult due to high levels of evolutionary divergence (6-8). Indeed, Ig receptor genes are completely absent from the genomes of invertebrates and jawless vertebrates [from which jawed vertebrates diverged some 500 million years ago and which have developed an alternative means of adaptive immunity bas...

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Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography–multiangle laser light scattering

Cited by 92 publications

References 28 publications

Protein aggregation: Pathways, induction factors and analysis

Protein aggregation: Pathways, induction factors and analysis

Characterization of monoclonal antibody size variants containing extra light chains

Structural reconstruction of protein ancestry

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