Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland.

The silkworm silk glands are powerful secretory organs that can produce and secrete proteins at high levels. As such, it has been suggested that the biosynthetic and secretory power of the silk gland can be harnessed to produce and secrete recombinant proteins in tight or loose association with silk fibers. However, the utility of the silkworm platform is constrained by the fact that it has a relatively primitive protein N-glycosylation pathway, which produces relatively simple insect-type, rather than mammalian-type N-glycans. In this study, we demonstrate for the first time that the silk gland protein N-glycosylation pathway can be glycoengineered. We accomplished this by using a dual piggyBac vector encoding two distinct mammalian glycosyltransferases under the transcriptional control of a posterior silk gland (PSG)-specific promoter. Both mammalian transgenes were expressed and each mammalian N-glycan processing activity was induced in transformed silkworm PSGs. In addition, the transgenic animals produced endogenous glycoproteins containing significant proportions of mammalian-type, terminally galactosylated N-glycans, while the parental animals produced none. This demonstration of the ability to glycoengineer the silkworm extends its potential utility as a recombinant protein production platform.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties.

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Expression of recombinant cyclooxygenase 1 in Drosophila melanogaster S2 cells transformed with human beta1,4-galactosyltransferase and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase.

We examined the expression of human cyclooxygenase-1 (COX-1) in Drososphila melanogaster S2 (S2) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (ST). Southern blot analysis indicated that multiple copies of the glycosyltransferases genes were integrated into the S2 cell genome. A lectin blot analysis also indicated that recombinant COX-1 from S2COX-1/GalT-ST cells contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. The specific peroxidase activity of recombinant sialylated COX-1 from S2COX-1/GalT-ST cells was 41,250 U mg(-1), indicating an increase of approximately 22% compared with a non-sialylated control (33,850 U mg(-1)) from S2COX-1 cells.

Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells.

We describe the expression and in vitro activity of recombinant canstatin from stably transformed Drosophila melanogaster S2 cells. Southern blot analysis indicated that transformed S2 cells contained multiple copies of the canstatin gene in the genome. Recombinant canstatin with a molecular weight of 29kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni(2+) affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED(50)) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37mug/ml. A maximum production level of 76mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5mM CuSO(4).

Characterization of a silkworm thioredoxin peroxidase that is induced by external temperature stimulus and viral infection.

A thioredoxin peroxidase (TPx) that reduces H(2)O(2) was firstly characterized in the lepidopteran insect, silkworm Bombyx mori. The B. mori TPx (BmTPx) cDNA contains an open reading frame of 585 bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BmTPx cDNA showed 78% identity to Drosophila melanogaster (DmTPx-1), 73% to Aedes aegypti (AaTPx), and 54-48% to other insect 2-Cys TPx. The cDNA encoding BmTPx was expressed as a 25-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BmTPx was shown to reduce H(2)O(2) in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor. Northern blot analysis revealed the presence of BmTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BmTPx in the fat body and midgut, but not in the hemolymph, suggesting the BmTPx is not secretable. When H(2)O(2) was injected into body cavity of B. mori larva, BmTPx mRNA expression was up-regulated in the fat body tissues. Interestingly, the expression levels of BmTPx enzyme in the fat body were particularly high when B. mori larva was exposed at low (4 degrees C) and high (37 degrees C) temperatures or baculovirus infection, suggesting that the BmTPx seems to play a protective role against oxidative stress caused by temperature stimuli and viral infection.