ABSTRACT
The source of recombinant collagen is clean, and it has the advantages of flexible sequence design, high yield and high purity, so it has a wide application prospect as biomaterials in tissue engineering and other fields. However, how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials. Unnatural amino acid O-(2-bromoethyl)-tyrosine was incorporated into collagen by two-plasmid expression system. The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol/L IPTG and 0.06% arabinose at 25 °C for 24 hours. The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation in pH 9.0 NH4HCO3 buffer, which formed aggregates with the largest molecular size up to 1 micrometre. The results pave the way for the design of recombinant collagen biomaterials.
Subject(s)
Amino Acids , Biocompatible Materials , Collagen/genetics , SulfidesABSTRACT
Objective:To develop an unnatural amino acid-labelled HiD-Hin47 fusion protein as a novel carrier.Methods:Twenty versions of the fusion protein were designed, each of which contained a different amino acid site replaced by an azide-bearing amino acid, N6-(2-azidoethoxy) (carbonyl)-L-Lysine (NAEK). These fusion proteins were constructed, expressed and purified, and the yield were evaluated by SDS-PAGE. Based on the highest protein yield, which was approximately 70% of the wild-type yield, the fusion protein with the unnatural amino acid in E677 site was selected. The pneumococcal polysaccharides of serotype 3 (F3) and serotype 6B (F6B) were coupled to the selected fusion protein through a "click" reaction. The conjugates were purified and compared in animal studies with other F3 and F6B conjugates that were coupled to CRM197, tetanus toxoid (TT) and HiD by conventional methods.Results:The immunogenicity of F3 conjugate using HiD-Hin47 as carrier (F3-HiD-Hin47) was slightly better than that of other F3 conjugates. F6B-HiD-Hin47 was significantly better than F6B-TT and F6B-HiD in terms of immunogenicity, but showed no significant difference with F6B-CRM197.Conclusions:NAEK-labelled HiD-Hin47 had the potential as a carrier for pneumococcal polysaccharide conjugate vaccine and was worthy of further study.
ABSTRACT
Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.