Expression, Purification, and Characterization of Ras Protein (BmRas1) from Bombyx mori

The Ras subfamily is the member of small G proteins superfamily involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation, and survival. Bombyx mori Ras-like protein (BmRas1) may belong to the Ras subfamily. It contained an H-N-K-Ras-like domain. The BmRas1 mRNA consisted of 1459 bp. The open reading frame contained 579 bp, encoding 192 amino acids. The protein had such secondary structures as α-helices, extended strand, and random coil. BmRas1 was expressed successfully in E. coli BL21. The recombinant protein was purified with metal-chelating affinity chromatography. The GTPase activity of purified protein was determined by FeSO4-(NH4)2MoO4 assay. The results showed that purified recombinant protein had intrinsic activity of GTPase. High titer polyclonal antibodies were generated by New Zealand rabbit immunized with purified protein. The gene expression features of BmRas1 at different stages and in different organs of the fifth instar larvae were analyzed by Western blot. The results showed that BmRas1 was expressed highly in three development stages including egg, pupae, and adult, but low expression in larva. BmRas1 was expressed in these tissues including head, malpighian tubule, genital gland, and silk gland. The purified recombinant protein would be utilized to further function studies of BmRas1

Expression, Purification and Bioactivities Analysis of Recombinant Active Peptide from Shark Liver

The Active Peptide from Shark Liver (APSL) was expressed in E. coli BL21 cells. The cDNA encoding APSL protein was obtained from shark regenerated hepatic tissue by RT-PCR, then it was cloned in the pET-28a expression vector. The expressed fusion protein was purified by Ni-IDA affinity chromatography. SDS-PAGE and HPLC analysis showed the purity of the purified fusion protein was more than 98%. The recombinant APSL (rAPSL) was tested for its biological activity both in vitro, by its ability to improve the proliferation of SMMC7721 cells, and in vivo, by its significant protective effects against acute hepatic injury induced by CCl4 and AAP (acetaminophen) in mice. In addition, the rAPSL could decrease the blood glucose concentration of mice with diabetes mellitus induced by alloxan. Paraffin sections of mouse pancreas tissues showed that rAPSL (3 mg/kg) could effectively protect mouse islets from lesions induced by alloxan, which indicated its potential application in theoretical research and industry

Subcellular localization and expression analysis of the BmDSCLP protein from silkworm, Bombyx mori

Leucine-rich repeat (LRR) proteins play important roles in the transduction of cellular signals and activation of defense responses. By scanning the cDNA library of silkworm (Bombyx mori) pupae constructed in our laboratory, we identified a 1557 bp gene that encodes a protein homologous to the death-associated small cytoplasmic leucine-rich protein, which was named as BmDSCLP. The full-length gene (GenBank accession no. FJ602779) contained a 642 bp open reading frame (ORF) encoding 213 amino acid residues. The ORF of this gene was inserted into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid and the fusion protein was expressed in Escherichia coli BL21(DE3) cells. The fusion protein was purified by Ni-affinity chromatography and fast protein liquid chromatography (FPLC) and its size was then, determined by liquid chromatography-mass spectrometry (LC/MS/MS) and found to be 27.74 kD. Polyclonal antibodies were raised by subcutaneous injection of the recombinant protein into New Zealand white rabbits and the titer reached 1:12800. Analysis of the subcellular localization of the BmDSCLP protein revealed that, the protein was localized in both the cytoplasm and nucleus, but the amount in the former was slightly higher than that in the latter. In addition, real-time fluorescence quantification polymerase chain reaction studies were conducted to investigate BmDSCLP transcription at different developmental stages and in different tissues of the fifth instar larva. The results indicated that, BmDSCLP is widely transcribed in different stages and tissues of the silkworm. Analysis of stage-specific transcription patterns indicated that, the transcriptional level of BmDSCLP was highest in adults and lowest in eggs. Analysis of tissue-specific transcription patterns revealed that, the transcriptional level of BmDSCLP was highest in genital organs and lowest in silk glands. These results suggest that BmDSCLP plays important roles in the reproductive development of B. mori.Keywords: Bombyx mori, death-associated small cytoplasmic leucine-rich protein, prokaryotic expression, fluorescence quantification polymerase chain reactio

Subcellular Localization and RNA Interference of an RNA Methyltransferase Gene from Silkworm, Bombyx Mori

RNA methylation, which is a form of posttranscriptional modification, is catalyzed by S-adenosyl-L-methionone-dependent RNA methyltransterases (RNA MTases). We have identified a novel silkworm gene, BmRNAMTase, containing a 369-bp open reading frame that encodes a putative protein containing 122 amino acid residues and having a molecular weight of 13.88 kd. We expressed a recombinant His-tagged BmRNAMTase in E. coli BL21 (DE3), purified the fusion protein by metal-chelation affinity chromatography, and injected a New Zealand rabbit with the purified protein to generate anti-BmRNAMTase polyclonal antibodies. Immunohistochemistry revealed that BmRNAMTase is abundant in the cytoplasm of Bm5 cells. In addition, using RNA interference to reduce the intracellular activity and content of BmRNAMTase, we determined that this cytoplasmic RNA methyltransferase may be involved in preventing cell death in the silkworm

Characterization of the Gene BmEm4, a Homologue of Drosophila E(spl)m4, from the Silkworm, Bombyx mori

The Drosophila E(spl)m4 gene contains some highly conserved motifs (such as the Brd box, GY box, K box, and CAAC motif) in its 3′ untranslated region (3′ UTR). It was shown to be a microRNA target gene in Drosophila and to play an important role in the regulation of neurogenesis. We identified a homologue of the E(spl)m4 gene from Bombyx mori called BmEm4 and examined the expression patterns of BmEm4 mRNA and protein. There was a lack of correlation in the expression of the mRNA and protein between the different developmental stages, which raises the possibility of posttranscriptional regulation of the BmEm4 mRNA. Consistent with this idea is the finding that the 3′ UTR contains two putative binding sites for microRNAs. Moreover, given that the expression is the highest in the larval head, as confirmed by immunohistochemistry, we propose that BmEm4 may also be involved in the regulation of neurogenesis. Immunostaining indicated that BmEm4 is located primarily in the cytoplasm

Molecular Characterization and Tissue Localization of an F-Box Only Protein from Silkworm, Bombyx mori

The eukaryotic F-box protein family is characterized by an F-box motif that has been shown to be critical for the controlled degradation of regulatory proteins. We identified a gene encoding an F-box protein from a cDNA library of silkworm pupae, which has an ORF of 1821 bp, encoding a predicted 606 amino acids. Bioinformatic analysis on the amino acid sequence shows that BmFBXO21 has a low degree of similarity to proteins from other species, and may be related to the regulation of cell-cycle progression. We have detected the expression pattern of BmFBXO21 mRNA and protein and performed immunohistochemistry at three different levels. Expression was highest in the spinning stage, and in the tissues of head, epidermis, and genital organs

In Vivo Bioassay of Recombinant Human Growth Hormone Synthesized in B. mori Pupae

The human growth hormone (hGH) has been expressed in prokaryotic expression system with low bioactivity previously. Then the effective B. mori baculovirus system was employed to express hGH identical to mature hGH successfully in larvae, but the expression level was still limited. In this work, the hGH was expressed in B. mori pupae by baculovirus system. Quantification of recombinant hGH protein (BmrhGH) showed that the expression of BmrhGH reached the level of approximately 890 μg/mL pupae supernatant solution, which was five times more than the level using larvae. Furthermore, Animals were gavaged with BmrhGH at the dose of 4.5 mg/rat.day, and the body weight gain (BWG) of treated group had a significant difference (P < .01) compared with the control group. The other two parameters of liver weight and epiphyseal width were also found to be different between the two groups (P < .05). The results suggested that BmrhGH might be used as a protein drug by oral administration

Bioavailability of Orally Administered rhGM-CSF: A Single-Dose, Randomized, Open-Label, Two-Period Crossover Trial

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF). METHODS AND FINDINGS: Using a single-dose, randomized, open-label, two-period crossover clinical trial design, 19 healthy volunteers were orally administered with BmrhGM-CSF (8 microg/kg) and subcutaneously injected with rhGM-CSF (3.75 microg/kg) respectively. Serum samples were drawn at 0.0h, 0.5h ,0.75h,1.0h,1.5h,2.0h ,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The hGM-CSF serum concentrations were determined by ELISA. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Three volunteers were randomly selected from 15 orally administrated subjects with ELISA detectable values. Their serum samples at the 0.0h, 1.0h, 2.0h, 3.0h and 4.0h after the administrations were analyzed by Q-Trap MS/MS TOF. The different peaks were revealed by the spectrogram profile comparison of the 1.0h, 2.0h, 3.0h and 4.0h samples with that of the 0.0h sample, and further analyzed using both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%. The rhGM-CSF peptide sequences in the serum samples were detected by MS analysis, and their sizes ranging from 2,039 to 7,336 Da. CONCLUSIONS: The results demonstrated that the oral administered BmrhGM-CSF was absorbed into the blood. This study provides an approach for an oral administration of rhGM-CSF protein in clinical settings. TRIAL REGISTRATION: www.chictr.orgChiCTR-TRC-00000107