Engineering specific chemical modification sites into a collagen‐like protein from Streptococcus pyogenes

Abstract

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

Evaluation of PC12 Cells’ Proliferation, Adhesion and Migration with the Use of an Extracellular Matrix (CorMatrix) for Application in Neural Tissue Engineering

“…The source of ECM proteins may be various organs and tissues whose proteins are obtained by mechanical destruction, enzymatic digestion, chromatography and precipitation. By contrast, bacterial and eucaryotic cell lines are used to produce recombinant ECM proteins, such as fragments of human recombinant fibronectin [ 12 , 13 , 14 ], various types of collagen [ 15 , 16 ], human tropoelastin [ 17 , 18 ] and laminin [ 19 , 20 ]. Bioscaffolds obtained as a result of decellularization are more desired than modified ECM synthetic constructions, as they ensure outcomes which are more optimal and closer to the natural environment for cell growth and functioning [ 21 ].…”

Prokaryotic Collagen-Like Proteins as Novel Biomaterials

“…Although this generated stable hydrogels, there was limited control over crosslink density and hydrogel properties ( Peng, Glattauer, and Ramshaw 2017 ). To resolve this problem, researchers introduced peptide sequences with functional residues to create additional crosslinking sites and stabilize the hydrogel network ( Stoichevska et al, 2016 ; Stoichevska et al, 2017 ). Stoichevska et al proposed introducing chemically functional residues to Scl2 proteins and forming crosslinks via oxidation ( Stoichevska et al, 2016 ; Stoichevska et al, 2017 ).…”

“…To resolve this problem, researchers introduced peptide sequences with functional residues to create additional crosslinking sites and stabilize the hydrogel network ( Stoichevska et al, 2016 ; Stoichevska et al, 2017 ). Stoichevska et al proposed introducing chemically functional residues to Scl2 proteins and forming crosslinks via oxidation ( Stoichevska et al, 2016 ; Stoichevska et al, 2017 ). As shown in Figure 3A , cysteine or tyrosine residues were introduced to either or both C-terminal and N-terminal of the collagen-like domain.…”

Photocrosslinkable triple helical protein with enhanced higher‐order formation for biomaterial applications

“…Chemical cross‐linking using 1‐ethyl‐3‐(3‐dimethylamino propyl) carbodiimide (EDC) 9 and glutaraldehyde 15 resulted in water‐unstable sponges and exhibited constraints in precisely controlling crosslinking density, toxicity, and hydrogel properties, respectively. To resolve this problem, additional crosslinking sites were created by introducing sequences with functional residues 16 or functionalized with photo‐cross‐linkable sites without perturbing the structure of the protein 17 . In this line, our lab has previously reported on the hydrogel formation with functionalized CLP with hydroxyproline and L‐3,4‐dihydroxyphenylalanine (L‐DOPA) by genetic code expansion facilitated by genipin crosslinking.…”