Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli.
Biomacromolecules
; 23(4): 1557-1568, 2022 04 11.
Article
em En
| MEDLINE
| ID: mdl-35258298
ABSTRACT
Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (â¼70%) is being secreted freely into the extracellular medium, with an initial purity of â¼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Colágeno
/
Escherichia coli
Limite:
Animals
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article