Development of novel non-animal collagen and gelatine materials
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1
CSIRO, CSIRO Manufacturing, Australia
Introduction: Collagens are the major protein in animals, being the principle components of skin, bone, ligament, tendon and cartilage. Collagen is commercially significant, most recently in biomedical products. When denatured, it forms gelatine which also has a wide range of medical applications. However, animal derived products such as collagen and gelatin do not have universal acceptance. In some cases this is due to concerns over transmission of diseases, but more frequently is due to beliefs and lifestyle choices. Just over a decade ago, it was realised that certain microorganisms also contain collagen like proteins which have similarities with the animal proteins. There was one key difference – the non-animal collagens from microorganisms do not require secondary modification – hydroxylation of proline – in order to achieve good stability. Rather, they use specific amino acid sequences to achieve good stability. This difference is a significant advantage as it allows recombinant production of these novel collagens in high yield in an E. coli system. In this study, we have studied the production and applications of a range of non-animal collagens and structural variants based on these sequences.
Materials and Methods: DNA sequences were based on the globular and collagen-like domains of the bacterial collagen genes from Streptococcus pyogenes, Solibacter usitatus, Rhodopseudomonas palustis and Methylobacterium sp., found in the GenBank database. Sequences were synthesised commercially. Various gene constructs were cloned into E. coli and expressed as triple-helical proteins using pCold vectors. Expression was evaluated using shake flasks prior to production in fed-batch fermentors using defined media. Expressed protein was extracted by sonication and purified by a combination of precipitation and proteolysis treatments. Biological, biochemical and biophysical analyses followed standard procedures.
Results and Discussion: The present study has shown that a range of non-animal collagens with different molecular weights and isoelectric points can be expressed in high yield in E. coli using scalable fermentors and purified by approaches compatible with large scale production. All form stable triple-helical structures. The use of defined media and avoidance of any animal derived materials allows a fully non-animal collagen product. Non-animal collagens were fabricated into various formats including hydrogels, membranes and sponges. Modifications to the sequences allowed introduction of biological functions, including cell binding domains. This allowed the non-animal collagens to mimic animal collagens in functional tests. One key difference, however, is that animal collagens after thermal denaturation and cooling form readily into a gel (gelatine). For non-animal collagens, eg: from S. pyogenes, S. usitatus and others, this is not the case – there is no quick gel formation. Sequence modifications, however, have been shown to give property changes that enable non-animal gelatine formation, giving new materials for biomedical applications.
Conclusions: We have shown that novel non-animal collagens can be produced in excellent yield and subsequently purified by a simple process. We have also shown that structural and functional variations can be readily introduced in recombinant products, illustrating the versatility of the non-animal collagens to provide products that can be tailored and fabricated to meet biomaterial requirements.
Keywords:
protein,
Scaffold,
material design,
biomacromolecule
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
New Frontier Oral
Topic:
Synthetic scaffolds as extracellular matrices
Citation:
Werkmeister
J,
Peng
YY,
Stoichevska
V,
Howell
L,
Dumsday
GJ and
Ramshaw
JA
(2016). Development of novel non-animal collagen and gelatine materials.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.01349
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.
*
Correspondence:
Dr. Linda Howell, CSIRO, CSIRO Manufacturing, Clayton, Australia, linda.howell@csiro.au