Expression And Purification Of Tag-free Recombinant Proteins In Silkworm,Bombyx Mori

With the rapid development of modern biotechnology, silkworm, Bombyx mori, hasbecome a very important bioreactor for expression and production of recombinant proteins.Silkworm Bac-to-Bac baculovirus expression system is a very useful eukayotic expressionsystem, which has a capability of post-translational modifications and is widely used toexpress efficiently recombinant proteins in silkworm cell lines and their larvae. However,the downstream separation and purification of target proteins has been explored less than theupstream recombination and expression technology established well in this expressionsystem, which has been a bottleneck to the wide application of silkworm as a bioreactor.Therefore, it is essential to establish a high-through and large-scale purification system ofrecombinant proteins produced in silkworm, achieving the goal of the commercialapplication of silkworm as a bioreactor and creating more economic value.In this thesis, enhanced green fluorescent protein (EGFP) selected as a model protein, atag-free recombinant proteins expression and purification system that utilizes the silkwormlarvae as expression host was established firstly, which applied Profinity eXact tag intosilkworm Bac-to-Bac baculovirus expression system. Through the modification of theoriginal donor plasmid pFastBac1, SP-PET-pFastBac1, a modified donor plasmid thatcontains both the signal peptide of silkworm fibroin heavy chain and Profinity eXact tag andused for the secretory expression of recombinant proteins, and PET-pFastBac1, the othermodified one that only contains Profinity eXact tag and used for the unsecretory expressionof recombinant proteins, were constructed, respectively. After generation of recombinantbaculovirus containing EGFP gene, EGFP was expressed in BmN cultured celld andsilkworm larvae, respectively. It showed that most of the EGFP fusion protein carryingsignal peptide secreted into the extracullular liquid, but the one without signal peptidealmost stayed in the cells, while the expression amount of the secreted EGFP fusion proteinwas much higher than that of the unsecreted one. A tag-free recombinant EGFP with highpurity was purified from cell culture media by Q-Sepharose anion exchangechromatography and subsequent Profinity eXact affinity chromatograohy. The classicalthree-step purification stradegy, namely Q-Sepharose anion exchange chromatography,Profinity eXact affinity chromatography and Superdex75gel filtration, was uede to purifyEGFP from the silkworm larval hemolymph and a tag-free one with relatively high puritywas obtained, of which the purification fold with860.5and the recovery field with26.7%. Secondly, silkworm P25protein was expressed in silkworm larvae and a tag-freerecombinant P25with high purity was purified from the larval hemolymph, using theestablished tag-free recombinant proteins expression and purification system. Both thesecreted P25and the unsecreted one were expressed efficiently in silkworm fifth-instarlarvae. It showed that most of the secreted P25fusion protein secreted into the hemolymph,but the unsecreted one almost stayed in the cells, while the expression amount of thesecreted P25was much higher than that of the unsecreted one. Meanwhile, the secreted P25has a glycosylation modification, which was consistent with the prediction. Through SPcation exchange chromatography, Butyl-S hydrophobic interaction chromatography,Profinity eXact affinity chromatography and Mono Q anion exchange chromatography, atag-free recombinant P25with high purity was purified from the hemolymph.At last, silkworm fibroin light chain (Fib-L) was expressed in silkworm larvae and atag-free recombinant Fib-L with high purity was purified from the larval hemolymph, usingthe eatablished tag-free recombinant proteins expression and purification system. Both thesecreted Fib-L protein and the unsecreted one were expressed efficiently in silkwormfifth-instar larvae. It showed that most of the secreted Fib-L fusion protein secreted into thehemolymph, but the unsecreted one almost stayed in the cells, while the expression amountof the secreted Fib-L protein was much higher than that of the unsecreted one. Meanwhile,the secreted Fib-L protein probably had some unexpected post-translational modifications.Through Butyl hydrophobic interaction chromatography, Q anion exchange chromatography,Profinity eXact affinity chromatography and Superdex75gel filtration, a tag-freerecombinant Fib-L protein with high purity was purified from the hemolymph.