Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Nanofabrication of chitosan-based dressing to treat the infected wounds: in vitro and in vivo evaluations.

Aim: Here, an innovative kind of antibacterial nanocomposite film is developed by incorporating graphene oxide and zinc oxide into chitosan matrix. Materials & methods: Our dressing was fabricated using the solution casting method. Fourier transform infrared spectra and TGA-DTG clearly confirmed the structure of film dressing. Results & conclusion: Our results showed the tensile strength and elongation at the break of the films were 20.1 ± 0.7 MPa and 36 ± 10%, respectively. Our fabricated film could absorb at least three-times the fluid of its dry weight while being biocompatible, antibacterial, non-irritant and non-allergic. In addition, it accelerated the healing process of infected wounds by regulating epithelium thickness and the number of inflammatory cells, thus it may be useful for direct application to damaged infected wounds.

In this study, an innovative kind of antibacterial nanocomposite film is developed by incorporating graphene oxide and zinc oxide into chitosan matrix. Our antibacterial wound dressing was fabricated using the solution casting method. Our fabricated film could absorb at least three-times the fluid of its dry weight while being biocompatible, antibacterial, non-irritant and non-allergic. In addition, our film accelerated the healing process of infected wounds by regulating epithelium thickness and the number of inflammatory cells. thus it may be useful for direct application to damaged infected wounds.

Chitosan nanoparticle loaded by epidermal growth factor as a potential protein carrier for wound healing: In vitro and in vivo studies.

Epidermal growth factor (EGF) can be efficiently used in wound healing process; but the main obstacle of its clinical use is its susceptibility to proteolysis and maintaining its effective concentration in the site of action. In this study, chitosan nanoparticles containing EGF is formulated using a simple method to increase its stability in physiological pH as well as protect its biological activity and effectiveness in wound healing process. Nanoparticles with different ratios of chitosan/EGF were prepared and evaluated in vitro and in vivo. Obtained results showed nanoparticles with 2:1 ratio of chitosan/EGF were able to release 80% of encapsulated protein after 12 h. Cell proliferation study demonstrated that prepared nanoparticles could protect EGF functionality in physiological pH. In vivo results showed that nanoparticles with 2:1 ratio of chitosan/EGF could significantly accelerate the wound closure-rate, re-epithelialisation and collagen deposition. In conclusion, the designed nanoparticles in optimal ratio can be considered as a potential vehicle for EGF delivery to wounds with the aim of improving healing process.