Supplementary MaterialsSupplementary Information srep16323-s1. yields. Furthermore, the r-haFGF protein bioactively promoted
May 11, 2019
Supplementary MaterialsSupplementary Information srep16323-s1. yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons. Human acidic fibroblast growth factor (haFGF) is a soluble heparin-binding protein that belongs to the fibroblast growth factor (FGF) family1. It harbors a molecular weight of DRIP78 15.8?kDa with 140-amino-acid peptides and functions as a strong mitogen to stimulate the proliferation of many cell types of mesodermal, endodermal and neuroectodermal origin, and is accordingly thought to play an important role in regulating angiogenesis and neovascularization during development and wound repair. Thus the haFGF is highly valuable in research, diagnostics and angiogenic therapeutic applications. For example, haFGF is CP-673451 cost widely used clinically to promote the rehabilitation and reconstruction of blood vessels2,3, scalds, and wound healing and has therapeutic potential for cardiovascular disorders4. In addition, haFGF is also applied in cosmetics to maintain strong vitality in skin cells, equipoise pigment distribution and improve skin character5,6. However, the limited sources of haFGF make it difficult to meet demands for the large amounts required for both and applications. Thus there is an increasing interest in the cost effective and efficient production of recombinant FGFs for experimental and clinical applications. Over the past few decades, several expression systems including recombinant adeno-associated virus (rAAV)7, now possesses the huge ability to synthesize a large amount of silk proteins in its silk gland and secrete them as silk thread to build a cocoon, making it an ideal bioreactor mode organ for the mass production of valuable recombinant proteins16. The silk thread is composed of CP-673451 cost two types of silk protein, the major fibroin proteins, which are synthesized in the posterior silk gland cells and account for 70%C80% of the silk thread weight, and the hydrophilic glue sericin proteins, which are synthesized in the middle silk gland cells and account for 20%C30% of the silk thread weight17. The fibroin proteins consist of fibroin heavy chains (H-chains), fibroin light chains (L-chains), and fibrohexamerins with a molar ratio of 6:6:118. The sericin proteins are mainly encoded by (((transposon-mediated transgenic techniques in the silkworm and the first successful expression of recombinant human type III procollagen in cocoons of transgenic silkworms20,21, efforts have been made CP-673451 cost to explore the silk gland of silkworm to be an efficient system for foreign protein production. Two major expression systems, the fibroin and sericin expression systems, were developed and over ten foreign proteins, including model proteins, human or animal-derived pharmaceutical proteins and silk-based proteins, have been successfully expressed in transgenic silkworm silk glands using these expression systems in the past decade22,23,24,25,26,27,28. These results showed that the silk gland is considered to be a cost-effective, easy-scale-up and simply processed bioreactor system for pharmaceutical protein production and silk genetically engineered proteins with improved mechanical properties and new biofunctionalities. In this study, we successfully produced an r-haFGF protein with high efficiency and biological activity in the cocoons of transgenic silkworm using our previously developed sericin-1 expression system26. A strategy involving PIG jumpstarter-induced remobilization of the expression cassette to a safe harbor genomic locus for the efficient expression of CP-673451 cost the transgene was explored and applied to increase the yields of the r-haFGF in the cocoons. The r-haFGF was conveniently purified from cocoons using a simple protocol. Further investigations indicated that the purified r-haFGF provides the same stimulation of NIH/3T3 cell growth, proliferation and wound healing as a commercial haFGF standard. Our results show that the silk gland of silkworm CP-673451 cost combined with jumpstarter-mediated remobilization.