High-Strength Collagen-Based Composite Films Regulated by Water-Soluble Recombinant Spider Silk Proteins and Water Annealing.

Spider silk has attracted extensive attention in the development of high-performance tissue engineering materials because of its excellent physical properties, biocompatibility, and biodegradability. Although high-molecular-weight recombinant spider silk proteins can be obtained through metabolic engineering of host bacteria, the solubility of the recombinant protein products is always poor. Strong denaturants and organic solvents have thus had to be exploited for their dissolution, and this seriously limits the applications of recombinant spider silk protein-based composite biomaterials. Herein, through adjusting the temperature, ionic strength, and denaturation time during the refolding process, we successfully prepared water-soluble recombinant spider major ampullate spidroin 1 (sMaSp1) with different repeat modules (24mer, 48mer, 72mer, and 96mer). Then, MaSp1 was introduced into the collagen matrix for fabricating MaSp1-collagen composite films. The introduction of spider silk proteins was demonstrated to clearly alter the internal structure of the composite films and improve the mechanical properties of the collagen-based films and turn the opaque protein films into transparency ones. More interestingly, the composite film prepared with sMaSp1 exhibited better performance in mechanical strength and cell adhesion compared to that prepared with water-insoluble MaSp1 (pMaSp1), which might be attributed to the effect of the initial dissolved state of MaSp1 on the microstructure of composite films. Additionally, the molecular weight of MaSp1 was also shown to significantly influence the mechanical strength (enhanced to 1.1- to 2.3-fold) and cell adhesion of composite films, and 72mer of sMaSp1 showed the best physical properties with good bioactivity. This study provides a method to produce recombinant spider silk protein with excellent water solubility, making it possible to utilize this protein under environmentally benign, mild conditions. This paves the way for the application of recombinant spider silk proteins in the development of diverse composite biomaterials.

Enhanced anticoagulant activity of hirudin-i analogue co-expressed with arylsulfotransferase in periplasm of E. coli BL21(DE3).

Hirudin, a blood anticoagulant, is the most potent natural thrombin inhibitor of leech origin. Its application is limited because it is difficult to obtain abundant natural hirudin directly from the leech. Although some bioengineering methods can significantly increase the production of hirudin, the reduced efficacy of recombinant hirudin (rH) remains a critical shortcoming. The lack of sulfation of tyrosine 63 in rH is an important cause of its inadequate performance. This article is the first report of periplasmic co-expression of an rH-I analogue with arylsulfotransferase (ASST) in E. coli BL21(DE3). Co-expressed rH-I analogue with sulfate donor substrate (p-nitrophenyl sulfate potassium) showed anticoagulant (rabbit and goat serum) activity twice more than rH-I analogue expressed without ASST, indicating its potential periplasmic sulfation. Moreover, purified rH-I analogue showed above 4.5 times higher anticoagulant activity compared to therapeutic anti-thrombotic heparin (HE). At the same time, pH-dependent differential solubility was employed to purify rH analogues from fermentation broth, which is a simple, fast and inexpensive purification technology, and can potentially be used for larger scale purification. This will also greatly improve the application of rH in clinical treatment.

The promising indicators of the thermal and mechanical properties of collagen from bass and tilapia: synergistic effects of hydroxyproline and cysteine.

Collagen has been widely documented as one of the most promising and competitive biomaterials for tissue engineering and medical applications. However, the properties of collagen differ from one source to another. In the present study, type I collagen (COL-I) was extracted and purified from the skins of Japanese sea bass (Lateolabrax japonicus) and Nile tilapia (Oreochromis niloticus). Ultraviolet (UV) spectroscopy, Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE were performed to characterize both COL-Is. The denaturing temperature of bass collagen (BC) was observed to be 27.2 °C, and 35.3 °C for tilapia collagen (TC). The content of hydroxyproline was 13.4% in TC, which was similar to that in porcine collagen (PC, 13.6%) and higher than that in BC (10.3%), while the content of cysteine in TC (0.87%) was significantly higher than that in PC (0.04%) and BC (0.35%). After incubation at different temperatures for 9 h, more degraded collagen bands appeared in the BC hydrogel (BCH) group than in the TC hydrogel (TCH) group, indicating that TCH exhibited better thermal stability than BCH. The thermal stabilities of TCH and PC hydrogel (PCH) were similar. The compressive stress of TCH was up to 0.099 MPa, while it was 0.047 MPa for BCH and 0.003 MPa for PCH. These results demonstrated that the content of amino acids (especially hydroxyproline and cysteine) has a synergistic effect on the thermal and mechanical properties of BCH, TCH and PCH, which would be an indicator of the thermal and mechanical properties of collagen hydrogels in future studies.