title: Protein crystallization with microseed matrix screening: application to human germline antibody Fabs.
abstract: 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.
authors.affiliation: Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Galina
authors.initials: G
authors.lastname: Obmolova
authors.affiliation: Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Malia
authors.affiliation: Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Teplyakov
authors.affiliation: Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Raymond W
authors.initials: RW
authors.lastname: Sweet
authors.affiliation: Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Gilliland
mesh_terms: D003460:Crystallization; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D011506:Proteins / Q000737:chemistry*
chemical_list: D007140:Immunoglobulin Fab Fragments; D011506:Proteins
keywords: Fabs; automation; crystallization; microseed matrix screening; optimization; seeding
references.citation: Acta Crystallogr D Biol Crystallogr. 2008 Dec;64(Pt 12):1222-7
references.pmid: 19018098
references.citation: Curr Opin Struct Biol. 2000 Oct;10(5):558-63
references.pmid: 11042454
references.citation: Nat Struct Biol. 1994 Mar;1(3):195-7
references.pmid: 7656038
references.citation: Protein Eng Des Sel. 2011 Sep;24(9):737-41
references.pmid: 21536542
references.citation: Protein Expr Purif. 2009 Oct;67(2):182-9
references.pmid: 19442740
references.citation: J Struct Biol. 2003 Apr;142(1):218-31
references.pmid: 12718933
references.citation: J Mol Biol. 2010 Mar 26;397(2):385-96
references.pmid: 20114051
references.citation: J Biol Chem. 1979 Dec 10;254(23):12219-23
references.pmid: 500706
references.citation: J Mol Biol. 2001 Sep 28;312(4):591-5
references.pmid: 11575916
references.citation: J Mol Biol. 1978 Aug 25;123(4):539-55
references.pmid: 691056
references.citation: Mol Immunol. 2012 Jun;51(2):227-33
references.pmid: 22487721
references.citation: Science. 1988 Jan 22;239(4838):385-7
references.pmid: 17836869
references.citation: Methods Enzymol. 2003;368:45-76
references.pmid: 14674268
references.citation: Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):414-23
references.pmid: 15299395
references.citation: Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14532-5
references.pmid: 8962086
references.citation: Acta Crystallogr D Biol Crystallogr. 1999 May;55(Pt 5):988-93
references.pmid: 10216295
references.citation: Acta Crystallogr D Biol Crystallogr. 2007 Apr;63(Pt 4):550-4
references.pmid: 17372361
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Oct 1;67(Pt 10):1290-5
references.pmid: 22102050
references.citation: Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7):1343-6
references.pmid: 12832806
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Aug;66(Pt 8):927-33
references.pmid: 20693692
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jan 1;64(Pt 1):14-8
references.pmid: 18097093
references.citation: J Struct Biol. 2003 Apr;142(1):66-76
references.pmid: 12718920
references.citation: J Struct Biol. 2003 Apr;142(1):162-9
references.pmid: 12718928
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Mar;60(Pt 3):601-5
references.pmid: 14993707