Abstract
The silk gland of Bombyx mori (BmSG) has gained significant attention by dint of superior synthesis and secretion of proteins. However, the application of BmSG bioreactor is still a controversial issue because of low yields of recombinant proteins. Here, a 3057 bp full-length coding sequence of Hpl was designed and transformed into the silkworm genome, and then the mutant (Hpl/Hpl) with specific expression of Hpl in posterior BmSG (BmPSG) was obtained. In the mutants, the transcription level of Fib-L and P25, and corresponding encoding proteins, did not decrease. However, the mRNA level of Fib-H was reduced by 71.1%, and Fib-H protein in the secreted fibroin was decreased from 91.86% to 71.01%. The mRNA level of Hpl was 0.73% and 0.74% of Fib-H and Fib-L, respectively, while HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted silk protein. The exogenous protein was therefore very efficiently translated and secreted. Further analysis of differentially expressed gene (DEG) was carried out in the BmPSG cells and 891 DEGs were detected, of which 208 genes were related to protein metabolism. Reduced expression of endogenous silk proteins in the BmPSG could effectively improve the production efficiency of recombinant exogenous proteins.
Highlights
Bombyx mori is the main target insect of human domestication and feeding selection in China, India, Uzbekistan, Thailand, and Brazil, and the sericulture industry utilizes silkworms for the efficient production of silk proteins as an important economic source for more than 30 million households
BmSG was not recognized as a major recombinant protein production platform[3,4,5], it could be the perfect host system for exogenous protein production because transgenic silkworms can be efficiently produced using piggyBac vectors[6,7], recombinant protein production can be targeted to the BmSG with tissue specific promoters[8,9,10,11,12,13], and the BmSG is naturally equipped to assemble exogenous proteins into secreted silk proteins[14]
In our laboratory previous work, we found an interesting phenomenon through observing the degenerated BmSG cells during the process of silkworm pupation, when silk proteins synthesis had stopped, the BmPSG began to efficiently synthesize the reproductive storage protein 30 K41, suggesting that high-efficiency protein synthesis functions of the BmSG cells could be used for the synthesis of exogenous proteins, if the silk protein genes were knocked down or knocked out
Summary
Bombyx mori is the main target insect of human domestication and feeding selection in China, India, Uzbekistan, Thailand, and Brazil, and the sericulture industry utilizes silkworms for the efficient production of silk proteins as an important economic source for more than 30 million households. In our laboratory previous work, we found an interesting phenomenon through observing the degenerated BmSG cells during the process of silkworm pupation, when silk proteins synthesis had stopped, the BmPSG began to efficiently synthesize the reproductive storage protein 30 K41, suggesting that high-efficiency protein synthesis functions of the BmSG cells could be used for the synthesis of exogenous proteins, if the silk protein genes were knocked down or knocked out. Based on this objective, Wang and Nakagaki[42] successfully constructed a Fib-H deficiency system by knocking out the heavy chain gene (Fib-H) of silk fibroin. This study focused on two questions: 1) Can a silkworm BmPSG system be established in which Fib-H expression is down-regulated, while at the same time maintaining the synthesis and secretion of Fib-L and P25 and achieving efficient secretion of exogenous proteins? and 2) What are the characteristic changes in genome expression of the BmSG tissues which allow more efficient secretion of exogenous proteins?
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