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A simple cost-effective methodology for large-scale purification of recombinant non-animal collagens

Applied Microbiology and Biotechnology volume 98, pages 1807–1815 (2014)Cite this article

Abstract

Recently, a different class of collagen-like molecules has been identified in numerous bacteria. Initial studies have shown that these collagens are readily produced in Escherichia coli and they have been isolated and purified by various small-scale chromatography approaches. These collagens are non-cytotoxic, are non-immunogenic, and can be produced in much higher yields than mammalian collagens, making them potential new collagens for biomedical materials. One of the major drawbacks with large-scale fermentation of collagens has been appropriate scalable down-stream processing technologies. Like other collagens, the triple helical domains of bacterial collagens are particularly resistant to proteolysis. The present study describes the development and optimization of a simple, scalable procedure using a combination of acid precipitation of the E. coli host proteins, followed by proteolysis of residual host proteins to produce purified collagens in large scale without the use of chromatographic methods.

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References

  • An B, DesRochers TM, Qin G, Xia X, Thiagarajan G, Brodsky B, Kaplan DL (2013) The influence of specific binding of collagen–silk chimeras to silk biomaterials on hMSC behavior. Biomaterials 34:402–412

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Birkedal-Hansen H, Taylor RE, Bhown AS, Katz J, Lin HY, Wells BR (1985) Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form. J Biol Chem 260:16411–16417

    CAS  PubMed  Google Scholar 

  • Boydston JA, Chen P, Steichen CT, Turnbough CL (2005) Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis. J Bacteriol 187:5310–5317

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Brodsky B, Ramshaw JAM (1997) The collagen triple-helix structure. Matrix Biol 15:545–554

    CAS  PubMed  Article  Google Scholar 

  • Cosgriff-Hernandez E, Hahn MS, Russell B, Wilems T, Munoz-Pinto D, Browning MB, Rivera J, Höök M (2010) Bioactive hydrogels based on designer collagens. Acta Biomater 6:3969–3977

    CAS  PubMed  Article  Google Scholar 

  • Ghosh N, McKillop TJ, Jowitt TA, Howard M, Davies H, Holmes DF, Roberts IS, Bella J (2012) Collagen-like proteins in pathogenic E. coli strains. PLoS One 7:e37872

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Kuhn K, Hannig K, Hörmann H (1961) Die Einwirkung von Trypsin und Pepsin auf natives Kollagen. Das Leder 10:237–241

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  PubMed  Article  Google Scholar 

  • Lyons RE, Lesieur E, Kim M, Wong DC, Huson MG, Nairn KM, Brownlee AG, Pearson RD, Elvin CM (2007) Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method. Protein Eng Des Sel 20:25–32

    CAS  PubMed  Article  Google Scholar 

  • Miller EJ, Rhodes RK (1982) Preparation and characterization of the different types of collagen. Methods Enzymol 82:33–64

    CAS  PubMed  Article  Google Scholar 

  • Mohs A, Silva T, Yoshida T, Amin R, Lukomski S, Inouye M, Brodsky B (2007) Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline. J Biol Chem 282:29757–29765

    CAS  PubMed  Article  Google Scholar 

  • Peng YY, Yoshizumi A, Danon SJ, Glattauer V, Prokopenko O, Mirochnitchenko O, Yu Z, Inouye M, Werkmeister JA, Brodsky B, Ramshaw JAM (2010) A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial. Biomaterials 31:2755–2761

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Peng YY, Howell L, Stoichevska V, Werkmeister JA, Dumsday GJ, Ramshaw JAM (2012) Pilot production of a collagen-like protein from Streptococcus pyogenes for biomedical applications. Microbial Cell Fact 11:146

    CAS  Article  Google Scholar 

  • Peng YY, Stoichevska V, Schacht K, Werkmeister JA, Ramshaw JAM (2014) Engineering multiple biological functional motifs into a blank collagen-like protein template from Streptococcus pyogenes. J Biomed Mater Res. doi:10.1002/jbm.a.34898

  • Persikov AV, Ramshaw JAM, Kirkpatrick A, Brodsky B (2005) Electrostatic interactions involving lysine make major contributions to collagen triple-helix stability. Biochemistry 44:1414–1422

    CAS  PubMed  Article  Google Scholar 

  • Pizarro-Guajardo M, Olguín-Araneda V, Barra-Carrasco J, Brito-Silva C, Sarker MR, Paredes-Sabja D (2014) Characterization of the collagen-like exosporium protein, BclA1, of Clostridium difficile spores. Anaerobe 25:18–30

  • Ramshaw JAM, Werkmeister JA, Glattauer V (1996) Collagen-based biomaterials. Biotechnol Genet Eng Rev 13:335–382

    CAS  PubMed  Article  Google Scholar 

  • Ramshaw JAM, Peng YY, Glattauer V, Werkmeister JA (2009) Collagens as biomaterials. J Mat Res Mat Med 20:S3–S8

    CAS  Article  Google Scholar 

  • Rasmussen M, Jacobsson M, Björck L (2003) Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins. J Biol Chem 278:32313–32316

    CAS  PubMed  Article  Google Scholar 

  • Ricard-Blum S (2011) The collagen family. Cold Spring Harb Perspect Biol 3:a004978

    PubMed  Article  Google Scholar 

  • Rosenbloom J, Harsch M, Jimenez S (1973) Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Arch Biochem Biophys 158:478–484

    CAS  PubMed  Article  Google Scholar 

  • Stephens LJ, Werkmeister JA, Tebb TA, Ramshaw JAM (1991) Identification of type III collagen from kangaroo skin. Das Leder 42:41–44

    CAS  Google Scholar 

  • Sylvestre P, Couture-Tosi E, Mock M (2002) A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. Mol Microbiol 45:169–178

    CAS  PubMed  Article  Google Scholar 

  • Vandersmissen L, De Buck E, Saels V, Coil DA, Anné J (2010) A Legionella pneumophila collagen-like protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells. FEMS Microbiol Lett 306:168–176

    CAS  PubMed  Article  Google Scholar 

  • Wang HM, Chan J, Pettigrew DW, Sodek J (1978) Cleavage of native type III collagen in the collagenase susceptible region by thermolysin. Biochim Biophys Acta 533:270–277

    CAS  PubMed  Article  Google Scholar 

  • Werkmeister JA, Ramshaw JAM (2000) Immunology of collagen-based biomaterials. In: Wise DL (ed) Handbook of biomaterials engineering. Marcel Dekker, New York, pp 739–759

    Google Scholar 

  • Werkmeister JA, Ramshaw JAM (2012) Recombinant protein scaffolds for tissue engineering. Biomed Mater 7:012002

    PubMed  Article  Google Scholar 

  • Xu Y, Keene DR, Bujnicki JM, Höök M, Lukomski S (2002) Streptococcal Scl1 and Scl2 proteins form collagen-like triple helices. J Biol Chem 277:27312–27318

    CAS  PubMed  Article  Google Scholar 

  • Xu C, Yu Z, Inouye M, Brodsky B, Mirochnitchenko O (2010) Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability. Biomacromolecules 11:348–356

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Yoshizumi A, Yu Z, Silva T, Thiagarajan G, Ramshaw JAM, Inouye M, Brodsky B (2009) Self-association of Streptococcus pyogenes collagen-like constructs into higher order structures. Protein Sci 18:1241–1251

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

This work was supported in part through NIH grant #EB011620. This study was facilitated by access to the Australian Proteome Analysis Facility supported under the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS).

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Affiliations

  1. CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, 3168, Australia

    Yong Y. Peng, Violet Stoichevska, Soren Madsen, Linda Howell, Geoff J. Dumsday, Jerome A. Werkmeister & John A. M. Ramshaw

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  1. Yong Y. Peng
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  2. Violet Stoichevska
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  3. Soren Madsen
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  4. Linda Howell
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  5. Geoff J. Dumsday
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  6. Jerome A. Werkmeister
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  7. John A. M. Ramshaw
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Correspondence to John A. M. Ramshaw.

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Peng, Y.Y., Stoichevska, V., Madsen, S. et al. A simple cost-effective methodology for large-scale purification of recombinant non-animal collagens. Appl Microbiol Biotechnol 98, 1807–1815 (2014). https://doi.org/10.1007/s00253-013-5475-8

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  • DOI: https://doi.org/10.1007/s00253-013-5475-8

Keywords

  • Non-animal collagen
  • Precipitation
  • Proteolysis
  • Purification
  • Triple helix