Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli

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 (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% 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.

A Review of Recent Progress on Collagen‐Based Biomaterials

“…[ 36 ] However, the occurrence of common diseases and related health problems in animals inhibits the production and use of animal proteins. [ 37 ] Artificial collagen fibrils can be obtained via self‐assembly and chemical synthesis to mimic some of the properties of natural collagen fibrils. [ 38 ] Moreover, it is possible to reduce the risk and uncertainty associated with animal‐derived products using genetic and fermentation engineering, while customizing desired peptides and optimizing the excellent properties of collagen.…”

Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial

“…112 The bacterial overexpression of the Scl2 protein was studied extensively and optimized for industrial-scale production, and subsequent downstream purifications methods. 113,114 The large-scale purification of the Scl2 protein can be achieved based on the recent advances in proteomics and chromatographic techniques. The recombinant Scl2 tagged with His-tag or StrepII tags could be easily purified using large-scale affinity-based chromatographic columns.…”

Bacterial collagen-templated synthesis and assembly of inorganic particles

“…Previously, an adapted secretion pathway, used natively by E. coli to secrete curli fibers, was reported for extracellular secretion of bacterial collagen (V ′ CL), which consists of the N-terminal variable domain plus N22, secretion signal peptide, (V ′ ), and a collagen domain (CL) [19]. We transformed an engineered plasmid (pET21d-v ′ cl-csgCEFG), containing the genes coding for V ′ Cl, CsgC, CsgE, CsgF, and CsgG, into E. coli PQN4 (a curli operon deletion mutant strain) for expression of bacterial collagen as the sole extracellular product.…”

“…We transformed an engineered plasmid (pET21d-v ′ cl-csgCEFG), containing the genes coding for V ′ Cl, CsgC, CsgE, CsgF, and CsgG, into E. coli PQN4 (a curli operon deletion mutant strain) for expression of bacterial collagen as the sole extracellular product. We then used a filtration-based purification method to isolate the extracellularly secreted collagen, as previously described [19]. Briefly, we digested the non-collagen impurities in the collected supernatant of the bacterial culture using pepsin (0.01 mg ml −1 , BioBasic, PB0688) and concentrated and isolated the bacterial collagen using crossflow filtration.…”

“…Previously, the extracellular secretion of bacterial collagen through a modified curli secretion pathway in E. coli was reported, and the purification methods were simplified for scalable isolation of bacterial collagen and subsequent fabrication of recombinant collagen-based biomaterials [19]. Although bacterial collagen is structurally similar to type I animal collagen, its potential for forming fibrillar structures is more limited compared with type I animal collagen [17].…”