Production of recombinant human epidermal growth factor fused with HaloTag protein and characterisation of its biological functions.

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.

Artificial ligament made from silk protein/Laponite hybrid fibers.

With developments in tissue engineering, artificial ligaments are expected to be future materials for anterior cruciate ligament (ACL) reconstruction. However, poor healing of the intraosseous part after ACL reconstruction significantly hinders their applications in this field. In this study, a bioactive clay Laponite (LAP) was introduced into the regenerated silk fibroin (RSF) spinning dope to produce functional RSF/LAP hybrid fibers by wet-spinning. These RSF/LAP hybrid fibers were then woven into artificial ligament for ACL reconstruction. The structure and mechanical properties of RSF/LAP hybrid fibers were extensively studied by different means. Results confirmed the presence of LAP in RSF fibers and revealed that the addition of LAP slightly deteriorated the comprehensive mechanical properties of RSF fibers. However, they were still much tougher (with higher breaking energy) than those of degummed natural silkworm silk that was earlier used for making artificial ligament. The artificial ligament woven from RSF/LAP hybrid fibers showed better cytocompatibility and osteogenic differentiation with mouse pre-osteoblasts in vitro than those made from degummed natural silkworm silks and pure RSF fibers. Furthermore, in vivo study in a rat ACL reconstruction model demonstrated that the presence of LAP in the artificial ligament could significantly enhance the graft osseointegration process and also improve the corresponding biomechanical properties of the artificial ligament. Based upon these results, the RSF/LAP hybrid fibers, which can be mass produced by wet-spinning process, are believed to have a great potential for use as artificial ligament materials for ACL reconstruction. STATEMENT OF SIGNIFICANCE: In this study, we successfully introduced Laponite (LAP), a kind of clay that has the function of osteogenic induction, into regenerated silk fibroin (RSF) fibers, which was prepared by a mature wet-spinning method developed in our lab. We believe that through artificial spinning, additional functional components can be added into RSF fibers, which one can hardly achieve with natural silks. We showed that the artificial ligament woven from RSF/LAP hybrid fibers had better cytocompatibility and osteogenic differentiation for mouse pre-osteoblasts in vitro, and significantly enhanced the graft osseointegration process and improved the corresponding biomechanical properties in a rat ACL reconstruction model in vivo, compared to those artificial ligaments made from degummed natural silkworm silks and pure RSF fibers.

Chitosan-based membrane chromatography for protein adsorption and separation.

A chitosan-based membrane chromatography was set up by using natural chitosan/carboxymethylchitosan (CS/CMCS) blend membrane as the matrix. The dynamic adsorption property for protein (lysozyme as model protein) was detailed discussed with the change in pore size of the membrane, the flow rate and the initial concentration of the feed solution, and the layer of membrane in membrane stack. The best dynamic adsorption capacity of lysozyme on the CS/CMCS membrane chromatography was found to be 15.3mg/mL under the optimal flow conditions. Moreover, the CS/CMCS membrane chromatography exhibited good repeatability and reusability with the desorption efficiency of ~90%. As an application, lysozyme and ovalbumin were successfully separated from their binary mixture through the CS/CMCS membrane chromatography. This implies that such a natural chitosan-based membrane chromatography may have great potential on the bioseparation field in the future.