Abstract
Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.
Highlights
Basic research aimed at developing clinical treatments for diseases has led to the production of a variety of medically useful proteins, including vaccines, growth factors, hormones, enzymes, and functional protein-based biomaterials
We report the expression of human epidermal growth factor (hEGF) in P25 gene knockout silkworms
Our results show that knockouts of the endogenous P25 protein can enhance the expression of hEGF under control of the P25 gene promoter
Summary
Basic research aimed at developing clinical treatments for diseases has led to the production of a variety of medically useful proteins, including vaccines, growth factors, hormones, enzymes, and functional protein-based biomaterials (which have recently become a research focus). FibH, FibL, and Ser gene promoters have been used to express a variety of proteins, including spider silk [8], feline interferon [9], human serum albumin [10], antibodies [11], and collagen [12], due to their strong activity. The P25 gene promoter is considered to have dual characteristics of a eukaryotic promoter and a silk gland specific promoter [13] and has been proven capable of driving the production of foreign proteins such as human insulin-like growth factor (hIGF-I) [14] and recombinant globulin [15]
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