Arthur Schveitzer Ferreira1,2, Amanda Lopacinski1,2, Michel Batista3, Priscila Mazzocchi Hiraiwa1, Natalia Fernanda Bueno4, Beatriz Gomes Guimarães1, Nilson I T Zanchin5,6. 1. Laboratory of Structural Biology and Protein Engineering, Carlos Chagas Institute, FIOCRUZ Paraná, Curitiba, PR, Brazil. 2. Cellular and Molecular Biology Graduate Program, Federal University of Paraná, Curitiba, PR, Brazil. 3. Mass Spectrometry Facility, Carlos Chagas Institute, FIOCRUZ Paraná, Curitiba, PR, Brazil. 4. Integrated Structural Biology Platform, Carlos Chagas Institute, FIOCRUZ Paraná, Curitiba, PR, Brazil. 5. Laboratory of Structural Biology and Protein Engineering, Carlos Chagas Institute, FIOCRUZ Paraná, Curitiba, PR, Brazil. nilson.zanchin@fiocruz.br. 6. Cellular and Molecular Biology Graduate Program, Federal University of Paraná, Curitiba, PR, Brazil. nilson.zanchin@fiocruz.br.
Abstract
BACKGROUND: Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. MATERIALS AND METHODS: The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. RESULTS: Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. CONCLUSION: The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.
BACKGROUND: Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. MATERIALS AND METHODS: The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. RESULTS: Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. CONCLUSION: The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.
Authors: D Zhang; M X Sliwkowski; M Mark; G Frantz; R Akita; Y Sun; K Hillan; C Crowley; J Brush; P J Godowski Journal: Proc Natl Acad Sci U S A Date: 1997-09-02 Impact factor: 11.205
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Authors: Wee-Tin Kao; Yanhong Wang; Joel E Kleinman; Barbara K Lipska; Thomas M Hyde; Daniel R Weinberger; Amanda J Law Journal: Proc Natl Acad Sci U S A Date: 2010-08-16 Impact factor: 11.205
Authors: D Meyer; T Yamaai; A Garratt; E Riethmacher-Sonnenberg; D Kane; L E Theill; C Birchmeier Journal: Development Date: 1997-09 Impact factor: 6.868