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J. Biol. Chem., Vol. 280, Issue 19, 18899-18907, May 13, 2005

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Synthesis of the Blood Circulating C-terminal Fragment of Insulin-like Growth Factor (IGF)-binding Protein-4 in Its Native Conformation

CRYSTALLIZATION, HEPARIN AND IGF BINDING, AND OSTEOGENIC ACTIVITY^*

Carlos Fernández-Tornero¶, Rosa M. Lozano, Germán Rivas, M. Ángeles Jiménez||, Ludger Ständker**, Diana Díaz-Gonzalez, Wolf-Georg Forssmann**, Pedro Cuevas, Antonio Romero, and Guillermo Giménez-Gallego

From the Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain, the ^||Instituto de Química Física Rocasolano, CSIC, 119 Serrano, Madrid 28006, Spain, ^**IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, Hannover D-30625, Germany, and the Servicio de Histología, Departamento de Investigación, Hospital Ramón y Cajal, Madrid 28034, Spain

Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an N-terminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein. The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional ¹H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

Received for publication, January 18, 2005 , and in revised form, February 23, 2005.

^* This work was supported in part by Grants BIO99-0867, BIO2001-1724, and BIO02-00305 from the Ministerio de Ciencia y Tecnología (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains four additional figures.

These two authors contributed equally to this work.

^¶ Supported by a Ph.D. fellowship from the Ministerio de Ciencia y Tecnología and by a grant of the Residencia de Estudiantes.

To whom correspondence should be addressed. Tel.: 34-91-8373112; Fax: 34-91-5360432; E-mail: gimenez_gallego{at}cib.csic.es.

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