Expression of Protein Complex Comprising the Human Prorenin and (Pro)Renin Receptor in Silkworm Larvae Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmids for Improving Biological Function
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- Du, D., Kato, T., Suzuki, F. et al. Mol Biotechnol (2009) 43: 154. doi:10.1007/s12033-009-9183-7
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Three forms of recombinant protein complexes comprising the human prorenin (hPro) and (pro)renin receptor (hPRR) (hPRR/prorenin) were successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids. They were localized in the fat body cells and formed a prorenin-bound hPRR complex. The expressed levels of hPro and hPRR were similar judging from Western blotting. The hPRR/prorenin complex containing 40 μg of hPRR (yield, 43%) and 30 μg of hPro (yield, 34%) was purified from 15 silkworm larvae by a series of purification using anti-FLAG and Strep-Tactin affinity chromatography. The renin activity of the purified hPRR/prorenin complex was 3.8-fold that of the mixture of hPRR and hPro expressed individually in vitro judging from the renin assay. These results show that the unstable transmembrane protein, hPRR, was coexpressed stably with ligand, hPro, and formed a stable protein, hPRR/prorenin complex that showed a high catalytic active form.
KeywordsSilkwormBombyx mori nucleopolyhedrovirusBacmid(Pro)renin receptorProrenin/(pro)renin receptor complex
Ligand–receptor complexes constituted by protein interaction appear to play a central role in most cellular functional pathways . The challenge for structural biology research is how to coexpress the ligands and receptors efficiently in the same host cell and purify them in order to obtain more functional information at the molecular level. Baculovirus-infected insect cells and mammalian systems present an attractive expression system for providing and performing most of complicated posttranslational processing of the proteins, and sometimes required for the ligand–receptor complex formation. This strategy can be better than the standard Escherichia coli system, although it is usually seen as time-consuming, expensive, and technically more challenging [2–4].
Nowadays silkworm larvae are used for expression system of eukaryotic proteins with complicate structure, because their protein expression level is 10- to 100-fold higher than that using insect cell. From these points of views, Bombyx mori nucleopolyhedrovirus (BmNPV)-infected B. mori silkworm larvae or pupae are the most suitable combination for large-scale productions of eukaryotic proteins. Unfortunately, however, the traditional preparation of recombinant baculovirus that express exogenous genes needs at least 40 days, because multiple rounds of purification and amplification of viruses are needed. Recently, the bacmid (a baculovirus shuttle vector) system had been developed for BmNPV . The BmNPV bacmid can be replicated in E. coli as a large plasmid and generate the recombinant virus DNA by the site-specific transposition in E. coli, and remains infectious with insect cells and silkworm larvae. Since this method eliminates multiple rounds of purification and amplification of virus, it greatly reduces both technical difficulty and time to select and purify recombinant viruses within 10 days. Therefore, the convenient bacmid system for BmNPV is using for as an important biotechnological method.
Renin–angiotensin system (RAS) plays an important role in the regulation of blood pressure; electrolyte balance; and is also involved in renal, neuronal, and endocrine functions related to cardiovascular control. Renin (EC 220.127.116.11) cleaves angiotensinogen to release the decapeptide angiotensin (ANG) I, which is further cleaved by ANG-converting enzyme to produce vasopressor peptide ANG II. Prorenin is the inactive precursor of renin, which has a prosegment with 43 amino acid residues attached at the N-terminus of renin. This prosegment has been considered to prevent interaction with angiotensinogen by covering the enzymatic cleft . A (pro)renin receptor (PRR) bound prorenin with higher affinity than renin and displayed ANG I-generating activity without proteolytic removal of the prosegment . Much physiological research has shown that the prorenin bound to PRR not only contributes to tissue ANG generation (particularly in subjects with diabetes), but also induces ANG-independent cardiovascular damage [8–10]. However, the interactions of prorenin with PRR and their structures have not been defined at the molecular level. Such efforts are critical to understand the molecular basis of ligand binding and for the ultimate goal toward the rational design of new receptor-blocking drugs. Insights into the three-dimensional structure of the receptor will facilitate the design of small ligands that can block PRR and be utilized therapeutically. Thus, the expression of the hPRR/prorenin complex will be highly significant for determination of its crystal structure.
Recently, we expressed the full-length hPRR successfully in insect cells and silkworm larvae and showed its proper localization in the functioning form [11, 12]. Here, we present a simple, cost effective, rapid method for the expression and purification of the protein complex comprising hPro and hPRR using silkworm larvae as a host. This approach provides a fast and affordable alternative strategy to produce mammalian proteins for structural and functional investigations. The hPro and hPRR were coexpressed stably in silkworm larvae and found to be formed as the protein hPRR/prorenin complex with a high biological active form.
Materials and Methods
Construction of Recombinant Prorenin and its Receptor Bacmids
Gene-specific primers for PCR
Coexpression and Localization of hPro and hPRR
Fifth-instar B. mori silkworm larvae (Ehime Sansyu, Co. Ltd., Yahatahama, Japan) were reared in a 25°C incubator and each was injected with 50 μl of bacmid solution. The bacmid solution consisted of 2.5 μg BmNPV-CPD/hPRR for (pro)renin receptor expression and 2.5 μg of one of the relevant bacmids (BmNPV-CPD/hPro, BmNPV-CPD/hPro-S, and BmNPV-CPD/hPro-RFP) for hPro expression, and 5 μl transfection reagent (DMRIE–C, Invitrogen) in 45 μl of PBS (8 g NaCl, 0.2 g KCl, 1.4 g Na2HPO4 · 2H2O, and 0.24 g KH2PO4 in 1 l of distilled water, pH 6.8). Half-an-hour after the injection, the larvae were fed with an artificial diet (Silkmate 2S, Nihon Nosan Co. Ltd., Yokohama, Japan) and reared for 6 days further. To investigate the localization of expressed proteins, the fat body was collected from silkworm larvae and immediately observed using a confocal laser scanning microscope (TCS-LS, Leica Microsystem, Heidelberg, Germany) equipped with an imaging system.
Preparation of Solubilized Microsomal Fraction from Fat Body Cells
The fat body collected at 6 days postinjection was sonicated in homogenization buffer (pH 7.4; Buffer A) comprising 50 mM sodium phosphate, 150 mM NaCl, and protease inhibitors (Roche, Lewes, Sussex, UK). Undisrupted cells and cellular debris were removed by centrifugation at 600×g for 10 min followed by a second centrifugation of the supernatant at 8,000×g for 30 min. The microsomal fraction was collected from the resulting pellet by centrifugation at 114,000×g for 60 min and was then washed twice with Buffer A. The pelleted microsomal fraction was resuspended in extraction buffer (50 mM sodium phosphate, 150 mM NaCl, protease inhibitors, and 1.5% dodecyl-β-d-maltoside (DDM), pH 8.0; Buffer B) to a final protein concentration of 5 mg/ml. Solubilized membrane proteins were collected from the supernatant after further centrifugation at 114,000×g for 60 min.
Coimmunoprecipitation assays were carried out using the Catch and Release version 2.0 Reversible Immunoprecipitation System (Millipore, Billerica, MA, USA). Five hundred micrograms of solubilized microsomal fraction and either 4 μg of anti-hPro or anti-FLAG antibody were loaded onto the spin columns containing 0.5 ml of immunoprecipitation capture resin for 1 h at 4°C with gentle agitation. Columns were washed three times, followed by sample elution. The eluates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation. Fluorescence analysis of GFPuv using a Molecular-FX multi-imager (Bio-Rad, Hercules, CA, USA), or Western blotting analysis using an anti-hPro antibody  that recognizes the hPro prosegment were performed. In additional negative control experiments, microsomal fractions containing single expressions of hPRR or hPro were used in the immunoprecipitation steps and each was detected with the antibody of its interacting partner.
Purification of Recombinant Complex Protein
Around 5 mg/ml of solubilized microsomal fraction was incubated in batches for 1.5 h at 4°C with 1/30 fraction volume of anti-FLAG M2 antibody agarose resin (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 1,000×g for 2 min and removal of the supernatant, the resin was loaded into a gravity flow column and washed with a 10-resin-volume buffer (50 mM sodium phosphate, 150 mM NaCl, protease inhibitors, and 0.2% DDM, pH 8.0; Buffer C). Proteins were eluted with a 5-resin-volume of Buffer C containing 100 µg/ml of FLAG peptide. The elution from the anti-Flag column containing the hPRR/prorenin complex was incubated for 1 h at 4°C with 300 µl of Strep-Tactin Superflow Resin (IBA GmbH, Göttingen, Germany). After loading the resin into gravity flow column and removing the flow-through, the resin was washed with a 3-resin-volume of Buffer C. Proteins were eluted in a 3-resin-volume of Buffer C containing 2.5 mM desthiobiotin.
SDS-PAGE and Western Blotting Analysis
Silkworm larvae at 6 days postinjection (d.p.i.) were bled by cutting the abdominal legs with scissors. Hemolymph was immediately mixed with 5 μl of 200 mM 1-phenyl-2-thiourea, centrifuged at 9000 rpm for 10 min at 4°C, and resulting supernatant was used for analysis. The larval fat body was isolated by dissection under sterile conditions at 4°C. It was then diluted with 25% (v/v) homogenization buffer (0.15-M NaCl, 2-mM EDTA, 2-mM dithiothreitol (DTT), and 20-mM Na2HPO4; pH 7.6) and sonicated on ice three times for 15 s each, with 15-s intervals, using a sonicator (VC 130PB, Sonic & Materials, Newtown, CT, USA). Finally, it was centrifuged at 13000 rpm for 30 min and resultant supernatant was used for analysis.
The fusion proteins contained in the hemolymph and fat body were detected by SDS-PAGE and Western blotting. SDS-PAGE was performed with 12% polyacrylamide gel using the Mini-PROTEAN II system (Bio-Rad). The respective bands were detected using a Molecular-FX multi-imager (Bio-Rad). For Western blotting, the samples were heated at 90°C for 10 min before running them on the SDS-PAGE gel. Mouse anti-FLAG-M2 as the primary antibody (1:20000, Sigma) and the anti-mouse IgG/HRP (horseradish peroxidase conjugated) antibody (1:20000, GE Healthcare, Buckinghamshire, UK) as the secondary antibody were used for detecting hPRR. Rabbit anti-hPro prosegment (1:5000) as primary antibody and the anti-rabbit IgG/HRP (1:20000, GE Healthcare) as a secondary antibody were used for detecting hPro. The immunoblot bands were visualized using enhanced chemoluminescence (ECL) plus Western blotting detection reagents (GE Healthcare) and analyzed using a Fluor-S/MAX multi-imager (Bio-Rad).
Assay of Renin Activity and Protein Concentration
Renin activity was measured using a SensoLyte 520 Renin Assay Kit (AnaSpec, Inc., San Jose, CA, USA). The 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) peptide is cleaved by renin and releases the fluorescence of 5-FAM, of which signal is monitored for the renin activity. Briefly, the purified samples were diluted to 50 μg/ml and seeded into a 96-well plate at 100 μl/well and incubated at 37°C for 30 min. At the same time, a 50 µl aliquot of the preincubated renin substrate solution containing 5-FAM/QXL 520 FRET peptide at 37°C was added to each well. After mixing the reagents completely by shaking the plate gently for 25 s, the released fluorescence signal of 5-FAM was measured at excitation/emission of 490/520 nm using Fluoromark (Bio-Rad). The fluorescence readings were represented as relative fluorescence units (RFU). The protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and bovine serum albumin as the standard.
Coexpression of hPro and hPRR in Silkworm Larvae
Localization of Human hPro and hPRR
To confirm the expression of protein complex of hPRR/prorenin, the solubilized microsomal fraction was prepared and subjected to coimmunoprecipitation with either anti-FLAG or anti-hPro antibodies. The eluates that were immunoprecipitated by anti-FLAG were also analyzed by Western blotting with anti-hPro antibody (Fig. 3d). Alternatively, the eluates that were immunoprecipitated by anti-hPro were analyzed by SDS-PAGE and visualized using a Molecular-FX multi-imager for detecting GFPuv fluorescence (Fig. 3e). The hPRR and hPro proteins were each specifically immunoprecipitated with the appropriate antibody, suggesting that the hPRR/prorenin complex was formed in the fat body by coexpression of hPro and hPRR.
Purification of the hPRR/Prorenin Complex
Purification of the hPRR/prorenin complex
Total proteina (mg)
133 ± 11
94 ± 08
89 ± 10
79 ± 03
67 ± 07
40 ± 05
30 ± 02
Functional Analysis of the hPRR/Prorenin Complex
Structure of extracellular domain of receptor usually has a binding affinity with the receptor’s ligand [16, 17]. Therefore, expression of the typically soluble extracellular domain of a receptor, rather than its full-length form, is employed to study the functions of a membrane protein. Through this approach, the use of detergents can be avoided and research on protein structure and function would be facilitated.
However, when the hPRR was expressed in silkworm larvae, it was located in the larval fat body, because of the hPRR composing of an extracellular domain, a transmembrane domain, and a cytoplasmic domain . We investigated the binding affinity of full length of hPRR, hPRR lacking cytoplasmic domain, and the extracellular domain of hPRR. Interestingly, the transmembrane domain of hPRR is indispensable in the formation of functional hPRR . The extracellular domain in the microsomal fraction of the fat body was observed to be bound with human renin while no affinity was observed after purification. However, when microsomal fraction of mock-injected fat body of silkworm larvae was added in the purified extracellular domain of hPRR, its binding affinity was recovered. It is probable that the purification of hPRR causes conformational change of extracellular domain of hPRR and an artificial transmembrane domain stabilizes the extracellular domain of hPRR. Mendrola et al.  reported that epidermal growth factor (ErB) receptor was stabilized by transmembrane domain interaction. Chiang and Knowles  reported that transmembrane domain interactions affected the stability of the extracellular domain of the human triphosphate diphosphohydrolase (NTPDase).
To avoid this unstable expression of hPRR, here, we expressed successfully the hPRR/prorenin complex using silkworm larvae and purified it efficiently. BmNPV/hPro and BmNPV/hPRR bacmids were coinjected to silkworm larvae, which made it possible to express the hPRR/prorenin complex stably. The expressed hPRR/prorenin complex was purified using two different kinds of affinity chromatography without contaminating hPRR and hPro. This approach provides a fast and affordable alternative strategy to produce stably mammalian proteins for structural and functional investigations. This is the first report on the coexpression and purification of an active hPRR/prorenin complex using a silkworm larva-based expression system. These results will assist in studying the structural interactions of prorenin and PRR.
In conclusion, the protein complex was expressed stably as a biologically active form and was purified with a high recovery. This coexpression system with two different kinds of bacmid is effective for the maintenance of protein stability and purification by forming a ligand–receptor complex. Moreover, this hPRR/prorenin complex might be useful for further crystallographic studies.
This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan.