Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

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Standard

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris. / Kjærgaard, Magnus; Gårdsvoll, Henrik; Hirschberg, Daniel; Nielbo, Steen; Mayasundari, Anand; Peterson, Cynthia B; Jansson, Anna; Jørgensen, Thomas J. D.; Poulsen, Flemming Martin; Ploug, Michael.

I: Protein Science, Bind 16, Nr. 9, 09.2007, s. 1934-45.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Kjærgaard, M, Gårdsvoll, H, Hirschberg, D, Nielbo, S, Mayasundari, A, Peterson, CB, Jansson, A, Jørgensen, TJD, Poulsen, FM & Ploug, M 2007, 'Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris', Protein Science, bind 16, nr. 9, s. 1934-45. https://doi.org/10.1110/ps.072949607

APA

Kjærgaard, M., Gårdsvoll, H., Hirschberg, D., Nielbo, S., Mayasundari, A., Peterson, C. B., Jansson, A., Jørgensen, T. J. D., Poulsen, F. M., & Ploug, M. (2007). Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris. Protein Science, 16(9), 1934-45. https://doi.org/10.1110/ps.072949607

CBE

Kjærgaard M, Gårdsvoll H, Hirschberg D, Nielbo S, Mayasundari A, Peterson CB, Jansson A, Jørgensen TJD, Poulsen FM, Ploug M. 2007. Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris. Protein Science. 16(9):1934-45. https://doi.org/10.1110/ps.072949607

MLA

Vancouver

Kjærgaard M, Gårdsvoll H, Hirschberg D, Nielbo S, Mayasundari A, Peterson CB o.a. Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris. Protein Science. 2007 sep;16(9):1934-45. https://doi.org/10.1110/ps.072949607

Author

Kjærgaard, Magnus ; Gårdsvoll, Henrik ; Hirschberg, Daniel ; Nielbo, Steen ; Mayasundari, Anand ; Peterson, Cynthia B ; Jansson, Anna ; Jørgensen, Thomas J. D. ; Poulsen, Flemming Martin ; Ploug, Michael. / Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris. I: Protein Science. 2007 ; Bind 16, Nr. 9. s. 1934-45.

Bibtex

@article{757cdf53a4ad4b18a88ba32324c3786c,
title = "Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris",
abstract = "The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.",
keywords = "Amides, Crystallography, X-Ray, Deuterium Exchange Measurement, Disulfides, Humans, Mass Spectrometry, Nuclear Magnetic Resonance, Biomolecular, Pichia, Plasminogen Activator Inhibitor 1, Protein Structure, Tertiary, Solutions, Somatomedins, Vitronectin",
author = "Magnus Kj{\ae}rgaard and Henrik G{\aa}rdsvoll and Daniel Hirschberg and Steen Nielbo and Anand Mayasundari and Peterson, {Cynthia B} and Anna Jansson and J{\o}rgensen, {Thomas J. D.} and Poulsen, {Flemming Martin} and Michael Ploug",
year = "2007",
month = sep,
doi = "10.1110/ps.072949607",
language = "English",
volume = "16",
pages = "1934--45",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Wiley-Blackwell Publishing, Inc.",
number = "9",

}

RIS

TY - JOUR

T1 - Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

AU - Kjærgaard, Magnus

AU - Gårdsvoll, Henrik

AU - Hirschberg, Daniel

AU - Nielbo, Steen

AU - Mayasundari, Anand

AU - Peterson, Cynthia B

AU - Jansson, Anna

AU - Jørgensen, Thomas J. D.

AU - Poulsen, Flemming Martin

AU - Ploug, Michael

PY - 2007/9

Y1 - 2007/9

N2 - The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.

AB - The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.

KW - Amides

KW - Crystallography, X-Ray

KW - Deuterium Exchange Measurement

KW - Disulfides

KW - Humans

KW - Mass Spectrometry

KW - Nuclear Magnetic Resonance, Biomolecular

KW - Pichia

KW - Plasminogen Activator Inhibitor 1

KW - Protein Structure, Tertiary

KW - Solutions

KW - Somatomedins

KW - Vitronectin

U2 - 10.1110/ps.072949607

DO - 10.1110/ps.072949607

M3 - Journal article

C2 - 17766387

VL - 16

SP - 1934

EP - 1945

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 9

ER -