Molecular Biotechnology

, Volume 43, Issue 2, pp 154–161

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

Authors

  • Dongning Du
    • Integrated Bioscience Section, Graduate School of Science and TechnologyShizuoka University
  • Tatsuya Kato
    • Faculty of AgricultureShizuoka University
  • Fumiaki Suzuki
    • Faculty of Applied Biological ScienceGifu University
    • Integrated Bioscience Section, Graduate School of Science and TechnologyShizuoka University
    • Faculty of AgricultureShizuoka University
Research

DOI: 10.1007/s12033-009-9183-7

Cite this article as:
Du, D., Kato, T., Suzuki, F. et al. Mol Biotechnol (2009) 43: 154. doi:10.1007/s12033-009-9183-7
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Abstract

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.

Keywords

SilkwormBombyx mori nucleopolyhedrovirusBacmid(Pro)renin receptorProrenin/(pro)renin receptor complex

Introduction

Ligand–receptor complexes constituted by protein interaction appear to play a central role in most cellular functional pathways [1]. 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 [24].

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 [5]. 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 3.4.23.15) 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 [6]. A (pro)renin receptor (PRR) bound prorenin with higher affinity than renin and displayed ANG I-generating activity without proteolytic removal of the prosegment [7]. 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 [810]. 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

The DNAs encoding human prorenin were amplified from a human kidney cDNA library (Clontech, Palo Alto, CA, USA) by polymerase chain reaction (PCR) using KOD polymerase and primers (Table 1). All PCR reactions were performed using the following program: 3 min at 94°C; 35 cycles at 94°C for 15 s, 50°C for 30 s, and 68°C for 90 s; followed by a final extension at 68°C for 5 min. The human prorenin (hPro) sequence was amplified using the forward primer (Primer 1) containing CACC and the native signal peptide sequences and the reverse primer (Primer 2) containing the related complementary sequence for encoding the C-terminal amino acids of hPro (Fig. 1). The Strep II-tagged hPro (hPro-S) sequence was amplified using the forward (Primer 1) and reverse primers (Primer 3) encoding the complementary sequence of enterokinase (EK) recognition and Strep II tag sites upstream to Primer 2 (Fig. 1). In order to monitor the hPro expression and its localization in vivo, a far-red fluorescent protein (RFP)-fused hPro was constructed. The RFP sequence was amplified from pHcRed1 (Clontech) using Primers 4 and 5. Primers 4 and 5 were introduced at Sph I and Sal I restriction enzyme cleavage sites upstream to Primers 1 and 2, respectively. The resulting fragment was digested with Sph I/Sal I and ligated to pHcRed1, which was also digested with the same enzymes (phPro-RFP). The resulting phPro-RFP was amplified using Primers 1 and 6 to obtain hPro-RFP. The amplified hPro, hPro-S, and hPro-RFP gene products were cloned into Gateway entry vectors using a pENTR/D-TOPO Cloning kit (Invitrogen, Carlsbad, CA, USA). After verification of the hPro DNA sequence, these entry clones were recombined with pDEST8 Gateway vector (Invitrogen) according to the manufacturer’s protocol. The generated plasmids were transformed into E. coli BmDH10Bac-competent cells containing the BmNPV-CPD bacmid [13], from which the cysteine protease gene had been deleted. The resulting transformed E. coli BmDH10Bac cells were grown on LB agar (10 g of bacto tryptone, 10 g of bacto yeast extract, 5 g of NaCl, and 15 g of agar per liter) plates containing 100 μg/ml of ampicillin, 50 μg/ml of kanamycin, 100 μg/ml of Blue-Gal, 7 μg/ml of gentamycin, 10 μg/ml of tetracycline, and 40 μg/ml of isopropyl-β-d-thiogalactopyranoside (IPTG). White antibiotics-resistant colonies were selected. Then, the BmNPV-CPD/hPro, BmNPV-CPD/hPro-S, and BmNPV-CPD/hPro-RFP bacmids were isolated from each transformed cells and their identities were confirmed by PCR using Primer 1 and each corresponding reverse primer. The practical BmNPV bacmid system [5] is directly applicable for the protein expression in silkworm. By using this system, target genes are introduced into the BmNPV bacmids by site-specific transposition and are under the control of polyhedrin promoter. The target protein can be successfully expressed in silkworm larvae and pupae not only by direct injection of its bacmid DNA but also by infection of its recombinant virus. Then The BmNPV-CPD/hPRR bacmid encoding (pro)renin receptor (hPRR) was prepared as described [11], to which bombyxin signal peptide, the UV-responsive green fluorescence protein (GFPuv), enterokinase (EK), and FLAG sites were fused in orderly sequence at the N-terminus of hPRR (Fig. 1).
Table 1

Gene-specific primers for PCR

Primer

Sequence

1

5′-CACCATGGATGGATGGAGAAGGATGC-3′

2

5′-TCAGCGGGCCAAGGCGAAGCCAATGCGGTTGTTACGCCGATCAAACTCTGTGTAG-3′

3

5′-TCACTTTTCGAACTGCGGGTGGCTCCACTTGTCGTCGTCATCGCGGGCCAAGGCGAAGCCAATGCGGTTG-3′

4

5′-TCAGCATGCACCATGGATGGATGGAGAAGGATG-3′

5

5′-TACGTCGACTTGTCGTCGTCATCGCGGGCCAAG-3′

6

5′-TCAGTTGGCCTTCTCGGGCAGGTCGCTG-3′

https://static-content.springer.com/image/art%3A10.1007%2Fs12033-009-9183-7/MediaObjects/12033_2009_9183_Fig1_HTML.gif
Fig. 1

Genetic construction of human prorenin (hPro), Strep II-tagged human prorenin (hPro-S), HcRed-fused human prorenin (hPro-RFP), and GFPuv-fused human (pro)renin receptor (GFPuv-hPRR)

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 Analysis

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 [14] 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.

Results

Coexpression of hPro and hPRR in Silkworm Larvae

When silkworm larvae were coinjected with BmNPV/hPRR and bacmids expressing hPro, hPRR expression was confirmed by observing the intensity of GFPuv under an ultraviolet illuminator (Fig. 2a). The observed green fluorescence image indicated that the GFPuv-hPRR fusion product was expressed in the fat body. Three forms of hPro were coexpressed with hPRR and analyzed by Western blotting analysis using an anti-hPro antibody that recognizes the prosegment region. Three types of recombinant hPro, hPro, hPro-S, and hPro-RFP, were detected with estimated molecular weights of 42, 44, and 70 kDa, respectively (Fig. 2b). However, the recombinant hPro was not detected in the hemolymph (Fig. 2c), indicating that hPro had accumulated specifically in fat body cells. A specific band of commercial renin (45 kDa) was not detected because it lacks the prosegment of hPro. This result revealed that the three forms of recombinant hPro were each expressed intact in the fat body of silkworm larvae. Combining together, these results indicate that hPRR and three forms of hPro were successfully coexpressed in silkworm larvae.
https://static-content.springer.com/image/art%3A10.1007%2Fs12033-009-9183-7/MediaObjects/12033_2009_9183_Fig2_HTML.gif
Fig. 2

Coexpression of recombinant hPRR and hPro in silkworm larvae. Coexpression of recombinant hPRR and hPro in silkworm larvae was confirmed by detecting GFPuv fluorescence under an ultraviolet illuminator (a). Western blotting analysis of hPro from homogenate of fat body (b) and hemolymph (c) using anti-hPro prosegment antibody. Fifty and fifteen micrograms of protein from homogenate of fat body and hemolymph were loaded, respectively. Lane 1, molecular weight marker; lanes 2 and 3, commercial prorenin and renin, respectively; lane 4, hPro; lane 5, hPro-S; lane 6, hPro-RFP; lane 7, mock (injection with a BmNPV bacmid alone)

Localization of Human hPro and hPRR

Equal weights of two recombinant BmNPV/hPRR and BmNPV/hPro-RFP bacmids were injected into the larvae and expressed proteins were observed using confocal laser scanning microscopy. Some of the cells only expressed hPro and some only expressed hPRR, but most expressed both hPro and hPRR (Fig. 3a–c). The hPRR was detected with GFPuv fluorescence (Fig. 3a), while hPro was also detected with red fluorescence (Fig. 3b). Two images were merged in yellow color (Fig. 3c), suggesting that two proteins were colocalized in fat body cells of silkworm larvae. In our previous report we demonstrated that hPRR fusion protein was localized in the microsomal fraction of fat body cells [11]. The expressed hPRR and hPro are assumed to be localized in the membranes of the fat body cells.
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Fig. 3

Colocalization and coimmunoprecipitation of hPRR and hPro. Colocalized hPRR/pro-RFP in fat body cells were observed by confocal laser scanning microscopy. hPRR (a) and hPro-RFP were detected with GFPuv fluorescence and HcRed fluorescence, respectively. Merging image of (a) and (b) is shown in (c). hPRR was immunoprecipitated with anti-FLAG antibody and followed by Western blotting with anti-hPro antibody (d). Prorenin was immunoprecipitated with anti-hPro antibody and followed by detection with GFPuv fluorescence (e). Lane M, fluorescence molecular weight maker; lane 1, hPro/hPRR complex; lane 2, hPro-S/hPRR complex; lane 3, hPro-RFP/hPRR complex. Closed and open arrows denote hPro and hPRR, respectively. Scale bar indicates 20 μm

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

The Strep II-tagged hPro and FLAG-tagged hPRR were coexpressed and purified by a series of purification using anti-Flag M2 antibody and Strep-Tactin superflow chromatography. This two-step affinity strategy can prevent contamination from hPro or hPRR proteins expressed individually in the purified protein complex during each affinity purification process. Solubilized microsomal fraction together with the nonionic detergent DDM was incubated with an anti-FLAG bearing agarose. The hPRR and hPRR/prorenin complex bound to the gel were eluted with FLAG peptide solution with recovery yield of 85% of hPRR and 75% of hPRR/prorenin complex, respectively (Table 2). The eluates were then incubated with Strep-Tactin resin and the hPRR/prorenin complex were eluted to yield the complex containing about 40 µg of hPRR and 30 µg of Strep II-tagged hPro, with recovery yields of 43% and 34%, respectively, This recovery of protein was obtained from 133 mg of solubilized microsomal protein extracted from 15 silkworm larvae (Table 2). The stoichiometric ratio of hPRR/prorenin was almost 1:1.
Table 2

Purification of the hPRR/prorenin complex

Purification stage

Total proteina (mg)

hPRR

hPro

(μg)b

Yield (%)

(μg)c

Yield (%)

Solubilized microsome

133 ± 11

94 ± 08

100

89 ± 10

100

Anti-Flag affinity

 

79 ± 03

85

67 ± 07

75

Strep-Tactin affinity

 

40 ± 05

43

30 ± 02

34

aTotal protein as determined by BCA protein assay kit

bhPRR determined by western blotting analysis using anti-Flag antibody with purified hPRR as a standard

chPro determined by western blotting analysis using anti-hPro antibody with purified hPro as a standard. Various amounts (5, 10, 25, 25, 50, and 100 ng) of purified hPRR and standard hPro with sample loading were analyzed by western blotting. Protein quantification was calculated by calibration curve correlated between intensity of immunoblot band visualized by ECL plus Western blotting detection reagent and its corresponding protein amount

Data represent mean ± standard deviation from three independent experiments

The elution fraction produced by anti-FLAG chromatography displayed a major band at 69 kDa by coomassie brilliant blue (CBB) staining (Lane 1, Fig. 4a). After further fractionation by Strep-Tactin affinity chromatography, two bands at 69 kDa and 44 kDa were detected (Lane 2, Fig. 4a). Western blotting analysis with anti-hPro and anti-FLAG antibodies revealed that the hPRR/prorenin complex appeared quite stable (Lanes 1 and 2, Fig. 4b).
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Fig. 4

Coomassie brilliant blue stained SDS-PAGE (a) and Western blotting (b) analysis of the purified hPRR/prorenin complex by FLAG and Strep-Tactin affinity resin. Lanes M, 1, and 2 denote molecular weight markers and eluates of FLAG affinity and of Strep-Tactin affinity, respectively. Closed arrows hPro, open arrows PRR

Functional Analysis of the hPRR/Prorenin Complex

It has been reported that the hPro bound with hPRR becomes fully active by a conformational change of the molecule after the prosegment is opened [7, 15]. Therefore, renin activity was measured to confirm the conformational change of the purified hPRR/prorenin complex. The hPRR/prorenin complex showed a 3.8-fold level of specific renin activity compared with a mixture of hPro and hPRR (Fig. 5). Catalytic rate of hPRR/prorenin complex was 161.7 RFU/min, while that of mixture of individually expressed hPro and hPRR was 41.7 RFU/min. hPRR and hPro alone did not show catalytic activity of renin, 7 RFU/min. This indicates that, when hPro and hPRR were coexpressed, the expressed hPro and hPRR in the fat body cells formed a hPRR/prorenin complex, which showed renin activity. Moreover, 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.
https://static-content.springer.com/image/art%3A10.1007%2Fs12033-009-9183-7/MediaObjects/12033_2009_9183_Fig5_HTML.gif
Fig. 5

Catalytic activity of hPRR/prorenin complex, hPRR, hPro, and mixed sample of hPRR and hPro. Protein sample (50 µg/ml) was used for measuring catalytic activity using a SensoLyte 520 Renin Assay Kit. closed circles hPRR/prorenin complex, closed triangles mixed hPRR and hPro, closed squares hPRR, open triangles hPro, open circles reaction buffer

Discussion

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 [11]. 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 [18]. 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. [19] reported that epidermal growth factor (ErB) receptor was stabilized by transmembrane domain interaction. Chiang and Knowles [20] 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.

Acknowledgments

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan.

Copyright information

© Humana Press 2009