Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

庚型肝炎病毒NS3蛋白在昆虫细胞中的表达

庚型肝炎病毒(HGV)/GB病毒C(GBV-C)疑似引起人类庚型肝炎[1～3].HGV和GBV-C为同一病毒的两个不同分离株,本文将其称为GBV-C/HGV.GBV-C/HGV属黄病毒科,为单股正链RNA病毒,全长约9.4kb.基因组中仅含有一个单一开放阅读框,编码E1、E2结构蛋白和NS2、NS3、NS4及NS5非结构蛋白.GBV-C/HGV的NS3蛋白具备丝氨酸蛋白酶活性和解旋酶活性[3],在NS3蛋白中还存在线性抗原表位[4],因此,NS3蛋白是GBV-C/HGV的重要功能蛋白.     

Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system

The recently cloned Na/P _i -cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P _i -cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K _m for P _i of 0.114 mm and an apparent K _m for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P _i -cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.We would like to thank W. Scherle and M. Lötscher (Institute of Anatomy) for their generous help using the confocal microscope and Ch. Gasser for the art work. Financial support by the Swiss National Fonds [Grant No. 32-30785.91 (to H.M.) and 32-28664.90 (to J.B.)] and by Stiftung für wissenschaftliche Forschung an der Universitát Zürich is greatly acknowledged.     

Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Functional Expression and Purification of Histidine-Tagged Rat Renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) Cotransporters

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P _i -cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/P _i -cotransport exhibited a K _m of P _i of 0.21 mm and an apparent K _m of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K _m of SO²⁻ ₄ of about 0.3–0.4 mm and a K _m of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS _i -1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography. Received: 24 March 1997/Revised: 8 July 1997     

High-Level Expression of Recombinant Active Human Renin in Sf-9 Cells: Rapid Purification and Characterization

Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%.     

Optimization of the production of full-length rCD4 in baculovirus-infected Sf9 cells

The baculovirus-insect cell system is reliable in expressing a variety of recombinant proteins. A recombinant baculovirus encoding the full length human CD4 has been used to infect Spodoptera frugiperda 9 cells in 6-L-airlift fermentors. The procedured described in this report permitted a 6.5-fold enhancement of rCD4 expression as compared to standard procedures previously published. The increase of rCD4 expression on the cell surface was achieved by using the following steps: (1) Optimal seeding density of 0.8 x 10(6) cells/mL used to multiply cells at a maximum exponential growth of 4.5 x 10(6); (2) high multiplicity of infection (MOI) of 580 PFU/cell; (3) addition of medium at time of infection. In addition to full-length rCD4, a "short" rCD4 with largely deleted cytoplasmic sequence (last 31 C-terminal amino acids) was also efficiently expressed.     

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.     

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.     

膜蛋白KCNN1在昆虫细胞Sf9中的表达与纯化

目的:利用昆虫细胞表达系统真核表达并纯化小电导钙激活钾离子通道蛋白1(KCNN1)。方法:以基因重组方法构建杆状病毒穿梭质粒reBacmid-KCNN1,将其转染至杆状病毒/Sf9细胞表达系统表达目的蛋白,并用Western印迹鉴定KCNN1的表达水平;用Ni-IDA-Sepharose CL-6B亲和层析柱纯化裂解细胞上清中的KCNN1,并用Western印迹鉴定纯化结果。结果:KCNN1在Sf9细胞中高效表达,通过亲和层析获得了纯化的KCNN1。结论:膜蛋白KCCN1在昆虫细胞Sf9中的表达与纯化,为深入研究其分子生物学功能提供了材料,也为全长膜蛋白的体外表达提供了一套可借鉴的实验方法。     

Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells

Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap.     

Characterization of the Functional Activity of Dopamine Ligands at Human Recombinant Dopamine D4 Receptors

Abstract: The human D₄ dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [³⁵S]GTPγS binding assay. By measuring D₄ receptor stimulation of [³⁵S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D₄ receptor, dopamine, quinpirole, and dp -2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [³⁵S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [³⁵S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D₄ receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [³H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.     

Multiple G<Subscript>i</Subscript> Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G_i/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gα_z or the PTX-resistant mutants of Gα_i1–3 and Gα_oA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G_i1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Characterization of Wild-Type and Amyotrophic Lateral Sclerosis-Related Mutant Cu,Zn-Superoxide Dismutases Overproduced in Baculovirus-Infected Insect Cells

Abstract: We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu²⁺ and Zn²⁺ were added to the medium. Cells grown in media supplemented with Cu²⁺ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu²⁺ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly⁴¹Asp, His⁴³Arg, and Gly⁸⁵Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.     

Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (I _K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but cells with I _K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (∼3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of I _K inactivation in 20 mm external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL I _K is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification,molecular cloning,and functional expression of inducible liver acylcarnitine hydrolase in C57BL/6 mouse,belonging to the carboxylesterase multigene family

To identify the peroxisome proliferator-inducible acylcarnitine hydrolase in C57BL/6 mice, acylcarnitine hydrolase was purified to homogeneity using column chromatography. The purified enzyme, named ACH M1, had a subunit molecular weight of 60kDa. ACH M1 could hydrolyze classical carboxylesterase (CES) substrates as well as palmitoyl-dl-carnitine and these activities were inhibited by anti-rat CES antibodies. The peptide fragments of ACH M1 were identical to those of the deduced amino acid sequence of mouse CES2 isozyme. These findings suggested that ACH M1 was a member of the CES2 family. The mouse CES2 cDNA, designated mCES2, was cloned from mouse liver. The recombinant mCES2 expressing in Sf9 cells showed high level of catalytic activity toward acylcarnitines. Furthermore, the biological characteristics of the expressed protein were identical with those of ACH M1 in many cases, suggesting that mCES2 encodes mouse liver ACH M1.