Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

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Free light chains and heavy/light chains pairs assay in diagnostics of multiple myeloma and other diseases related to plasmatic cells dyscrasias

Diagnostyka Laboratoryjna ◽

10.5604/01.3001.0013.7962 ◽

2017 ◽

Vol 53 (1) ◽

pp. 41-46

Author(s):

Ewelina Kudyba ◽

Tomasz Wróbel

Keyword(s):

Multiple Myeloma ◽

Residual Disease ◽

Protein Electrophoresis ◽

Light Chains ◽

Free Light Chains ◽

Heavy Chains ◽

Plasma Cell Neoplasms ◽

Single Clone ◽

Minimal Residual

Plasma cell neoplasms constitute a large group of diseases characterized by uncontrolled proliferation of a single clone of plasmocytes and production of monoclonal protein which may be present in patient’s serum in the form of intact immunoglobulins, free light immunoglobulin chains, or both of these molecules simultaneously. In addition to the methods commonly used for years for the determination of the protein such as protein electrophoresis or immunofixation, clinical standards in the last decade included the test for determining the concentration of κ and λ free light chains in serum. The test profile mentioned above has been complemented by a new method for identifying and determining the concentration of immunoglobulins with the possibility of recognizing the binding between pairs of heavy chains γ, α, μ and κ or λ light chains of immunoglobulins. It gives the opportunity to differentiate separately Ig’κ and Ig’λ molecules in each immunoglobulin class. Quantification of these sensitive and specific markers is used for the early diagnosis of the disease and it also provides the ability to accurately monitor the treatment, evaluate minimal residual disease and detect early the recurrence of monoclonal gammopathy like multiple myeloma.

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Immunochemical Characterization of a Monoclonal γG4, λ Human Antibody to Factor IX

Blood ◽

10.1182/blood.v40.1.1.1 ◽

1972 ◽

Vol 40 (1) ◽

pp. 1-10 ◽

Cited By ~ 19

Author(s):

Isadore M. Pike ◽

William J. Yount ◽

Elliot M. Puritz ◽

Harold R. Roberts

Keyword(s):

Gel Filtration ◽

Factor Ix ◽

Light Chains ◽

Human Antibody ◽

Inhibitor Activity ◽

Heavy Chains ◽

Human Factor Ix ◽

Zone Electrophoresis ◽

Kappa Light Chains

Abstract A circulating inhibitor specific for factor IX in a patient with hemophilia B was characterized with antisera to human immunoglobulins. Inhibitor-rich plasma was mixed with an excess of specific antiserum and then assayed for residual inhibitor activity. Inhibitor activity was completely removed by antisera specific for γG4 heavy chains and lambda light chains. Antisera specific for γA, γM, γD, γE, γG1, γG2, and γG3 heavy chains and kappa light chains had no effect on the inhibitor. On preparative zone electrophoresis, inhibitor activity was localized in a sharp band in the anodal portion of the γG peak, an electrophoretic distribution paralleling that of γG4. Precise localization of the inhibitor activity on calibrated gel filtration columns revealed a relatively narrow zone of fractions containing the inhibitor, wherein the proteins have an estimated molecular weight of 145,000. The relative homogeneity of the antibody may reflect specificity for a uniform, discrete portion of the human factor IX molecule.

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Protein Crystallization on Lipid Layers and Structure Determination of the RNA Polymerase II Transcription Initiation Complex

Journal of Biological Chemistry ◽

10.1074/jbc.274.11.6813 ◽

1999 ◽

Vol 274 (11) ◽

pp. 6813-6816 ◽

Cited By ~ 9

Author(s):

Francisco J. Asturias ◽

Roger D. Kornberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Structure Determination ◽

Transcription Initiation ◽

Protein Crystallization ◽

Initiation Complex ◽

Transcription Initiation Complex ◽

Lipid Layers ◽

Rna Polymerase Ii Transcription

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Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102869 ◽

1988 ◽

Vol 46 ◽

pp. 156-157

Author(s):

Donald A. Winkelmann

Keyword(s):

Electron Microscopy ◽

Molecular Weight ◽

Actin Filaments ◽

Light Chains ◽

Myosin Filament ◽

Primary Role ◽

Heavy Chains ◽

Myosin Subfragment ◽

Myosin Subfragment 1 ◽

Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

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