Abstract
BackgroundSilk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention.ResultsIn this study, we established a transgenic silkworm strain which synthesizes the recombinant human lactoferrin (rhLF) in the silk gland and spins them into the cocoon by our previously constructed silk gland based bioreactor system. The yield of the rhLF with the highest expression level was estimated to be 12.07 mg/g cocoon shell weight produced by the transgenic silkworm strain 34. Utilizing a simple purification protocol, 9.24 mg of the rhLF with recovery of 76.55% and purity of 95.45% on average could be purified from 1 g of the cocoons. The purified rhLF was detected with a secondary structure similar with the commercially purchased human lactoferrin. Eight types of N-glycans which dominated by the GlcNAc (4) Man (3) (61.15%) and the GlcNAc (3) Man (3) (17.98%) were identified at the three typical N-glycosylation sites of the rhLF. Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the Escherichia coli (E. coli) and Bacillus subtilis.ConclusionsThese results show that the middle silk gland of silkworm can be an efficient bioreactor for the mass production of rhLF and the potential application in anti-inflammation and antibacterial.
Highlights
Silk glands are used by silkworms to spin silk fibers for making their cocoons
200 nondiapause silkworm embryos were microinjected with a mixture of plasmid consisting the phSrhLFSer1 and helper vector, among which, 42.0% injected embryos were successfully hatched and were carefully fed until the moth stage to oviposit the G1 offspring
Expression of inflammatory nitric oxide synthase (iNOs) in LPS-induced macrophages was investigated after recombinant human lactoferrin (rhLF) treatment, the results showed that the expression
Summary
Silk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention. Human lactoferrin has various biological functions, in particular, widely known bactericidal and bacteriostatic activities that form the primary line of defense against the. Human lactoferrin has many other biological functions including antitumor [10], antioxidant [11] and stimulate the proliferation in many cell types [12]. Human lactoferrin is regarded as a potential compound for the food, cosmetics, and feed additives applications. Poor yields of recombinant protein, lack of post-translational modification, and a complex expression process has severely hindered these expression system applications
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