PDF
ePub
Citation
Print
Abstract
The Bombyx mori decapentaplegic gene is one of the conserved genes in vertebrate and invertebrates. The TGF-β superfamily contains conserved polypeptide growth factors that play important roles in different cellular processes such as proliferation, apoptosis, differentiation and cell-fate determination. The B. mori dpp gene shares genetic homology with hBMPs and Drosophila dpp. Until now, only few studies have been conducted to examine the functions of B. mori dpp; and hence, its function is not yet well understood. In this study, the baculovirus expression vector system (BEVS) was used for expression of the recombinant B. mori dpp protein and in which the recombinant baculovirus is recovered in the host Sf9 cells. The selected pure recombinant baculovirus containing B. mori dpp gene (rBV-egfp-Bm dpp) was used to increase the effective protein purification by using His-tag extraction strategy. After selection of recombinant baculovirus, recombinant B. mori dpp proteins were extracted from the re-infected cells with pure rBV-egfp-Bm dpp. Herein, we summarize the efficient expression and purification of B. mori dpp proteins from the insect cells using the BEVS. This recombinant protein could be suitable for functional test and various application studies.
REFERENCES
1). Chen Y, Riese MJ, Killinger MA, Hoffmann FM. A genetic screen for modifiers of Drosophila decapentaplegic signaling identifies mutations in punt, Mothers against dpp and the BMP-7 homologue, 60A. Development. 1998; 125:1759–68.
2). Künnapuu J, Shimmi O. Evolutional imprints on the sequences of BMP2/4/DPP type proteins. Fly (Austin). 2010; 4:21–3.
3). Park SW, Goo TW, Choi GH, Kang SW, Kim SW, Kim SR. Functional analysis of Bombyx mori Decapentaplegic gene for bone differentiation in a mammalian cell. Int J Indust Entomol. 2013; 27:159–65.
4). Park SW, Lee HK, Kim TG, Yoon SK, Paik SY. Hepatocyte-Specific Gene Expression by Baculovirus Pseudotyped with Vesicular Stomatitis Virus Envelope Glycoprotein. Biochem Biophys Res Commun. 2001; 289:444–50.
5). Jin JY, Park CJ, Park SW, Paik SY. Protein Expression of the Human Norovirus Capsid Gene using the Baculo-virus Expression System. J Bacteriol Virol. 2011; 41:183–7.
6). Russell D, Sambrook J. Molecular cloning: a laboratory manual. 3rd ed.Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press;2000.
7). Sampath TK, Rashka KE, Doctor JS, Tucker RF, Hoffmann FM. Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals. Proc Natl Acad Sci U S A. 1993; 90:6004–8.
Figure 1.
Cloning of the Bombyx mori dpp expression vectors. The 1,324-bp full-length cDNA of B. mori dpp was synthesized using total RNA obtained from fat bodies of 3-day-old of 5th instar larvae. B. mori dpp gene cassette inserted into the pBACgus4X-egfp plasmid DNA with Bam H I and Not I restriction enzymes is terminated by stop codon of vector and added 6X His amino acids to 3′-end region. pBACgus4X-egfp vector contain the 0.7-kb EGFP downstream of the other p10 promoter. This fragment was inserted into the pBAC-gus4X-egfp vector in a direct orientation with respect to the polh promoter to generate the pBAC-gus4X-Bm dpp plasmid DNA.
Figure 2.
Genomic DNA PCR analysis of Sf9 cells transfected with the rBV-egfp or rBV-egfp-Bm dpp recombinant viruses.(A) To evaluate dpp expression in cells transfected with plasmid DNA, we performed genomic DNA PCR analysis by using a specific primer pair. An approximately 500-bp egfp gene PCR fragment was amplified from the genomic DNA of Sf9 cells infected with rBV-egfp or rBV-egfp-Bm dpp recombinant viruses. (B) An approximately 213-bp B. mori dpp gene PCR fragment was amplified from the genomic DNA of Sf9 cells infected with rBV-egfp or rBV-egfp-Bm dpp recombinant viruses. 1: positive control (plasmid DNA); 2: negative control; 3. Sf9 cells infected without rBV; 4: Sf9 cells infected with rBV-egfp; 5. Sf9 cells infected with rBV-egfp-Bm dpp.
Figure 3.
RT-PCR analysis of dpp expression with RNA extracted from different cells of rBV infected and uninfected. To determine whether the B. mori dpp gene was expressed in the target cells, we performed RT-PCR analysis. A 213-bp fragment of the B. mori dpp gene was amplified from only rBV-egfp-Bm dpp recombinant virus infected sample that was transfected with the pBAC-gus4X-Bm dpp plasmid DNA. 1: positive control (plasmid DNA); 2: negative control; 3. Sf9 cells infected without rBV; 4: Sf9 cells infected with rBV-egfp; 5. Sf9 cells infected with rBV-egfp-Bm dpp.
Figure 4.
Western blot analysis of B. mori dpp protein induction in Sf9 cells infected with the rBV-egfp-Bm dpp recombinant baculovirus. To confirm of recombinant B. mori dpp protein purification condition, selected recombinant rBV-egfp-Bm dpp from A was infected in Sf9 cells and harvested at 72 h post infection. The specificity of each antibody is indicated each rBV-egfp-Bm dpp with B. mori dpp gene. And positions and molecular weights of protein size markers are indicated on the left (37 kDa; red bar).
XML Download