Abstract
BackgroundSafe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3′-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination.ResultsThe fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2 × 2 mm bleeding wound in the mouse liver after 27.21 ± 1.92 s, 18.65 ± 1.97 s and 15.85 ± 1.21 s, respectively. The hemostasis time was 21.23 ± 1.84 s for rat-tail collagen and 14.44 ± 1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64 ± 1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze.ConclusionsThese results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.
Highlights
Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage
Construction of clone vectors To achieve some novel hemostatic biomaterials, several fusion proteins were designed based on self-assembling peptide human elastin-like polypeptides fusion RADA-16 (RADA-16) and hELPs with inverse phase transition cycles (ITC) property (Fig. 1a)
RADA-16 coding sequences composed of optimized codons in accordance with the codon bias of E. coli were fused to the 3′-end of the open reading frame (ORF) of hELP36, hELP60 and hELP96 using the restriction endonuclease recognition sites of BamHI at 5′-end and XhoI at the 3′-end of RADA-16-encoding sequence, as well as the BamHI &
Summary
Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In the event of hemorrhage, natural hemostasis is slow and complicated, including many processes such as vasal contraction, platelet aggregation and progressive activation of coagulation factors. Polysaccharide hemostatic materials including chitosan, starch and cellulose can rapidly adhere to wounds and absorb moisture, accelerate the formation of blood clots and stop bleeding. Another prominent protein-based hemostatic material is fibrin glue, which contains thrombin and can effectively accelerate the activation of coagulation factors to stop bleeding [6]. Fusion protein strategy provide a way to improve the stability of small MW peptide [15]
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