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.     

Secretion of the extracellular domain of the human insulin receptor from insect cells by use of a baculovirus vector.

To explore the utility of the baculovirus/insect-cell system for the expression of a soluble secreted human insulin-receptor (hIR) extracellular ligand-binding domain, we have engineered a recombinant virus encoding an hIR deletion mutant which is truncated eight residues from the beginning of the predicted transmembrane domain (i.e. 921 residues). Within 24 h after infection of Sf9 cells with virus, insulin-binding activity begins to accumulate in the culture medium, and reaches a maximum between 48 and 72 h. The intracellular transit and processing of this secreted receptor, designated 'AchIR01', is quite slow. After 24 h in pulse-chase experiments approximately 50% of the metabolically labelled protein is still inside the cell. This protein accumulates as a non-cleaved hIR precursor which is glycosylated, but the carbohydrate is entirely endoglycosidase H (endoH)-sensitive (i.e. high mannose). Approximately one-half of the receptor in the culture medium (i.e. approximately 25% of the total) is in the form of non-cleaved precursor, and about one half of its carbohydrate chains are now endoH-resistant. The remainder of the protein is proteolytically processed hIR (alpha-plus truncated beta-subunits). None of these hIR species exhibit O-linked carbohydrate. Only the processed form of the receptor in the medium binds insulin. This insulin-binding protein is secreted as a dimer (alpha beta)2, and binds insulin with an affinity which is comparable with that of both the wild-type hIR as well as the secreted form of the hIR expressed in mammalian cells. Despite the rather inefficient processing and altered glycosylation of the AchIR01 protein in insect cells, this high-affinity insulin-binding protein accumulates in the medium at levels (mg/litre) of about 100 times that achieved in a mammalian-cell system.     

Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin.

Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.     

Existence of catalase-less peroxisomes in Sf21 insect cells

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.     

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.     

Functional expression and characterization of dipeptidyl peptidase IV from the black-bellied hornet Vespa basalis in Sf21 insect cells

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.     

Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λ_max = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.     

Expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.     

Characterization of cathepsin L secreted by Sf21 insect cells

Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed.     

Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.     

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.     

昆虫细胞制备AAV-ITR基因表达微载体

AAV-ITR基因表达微载体是只含有腺相关病毒(Adeno-associated virus,AAV)倒置末端重复序列(Inverted terminal repeats,ITR)、基因表达顺式元件和目的基因,而不含有其他外源DNA序列的双链或单链DNA。本研究利用杆状病毒表达系统,制备得到两种重组杆状病毒Bac-ITR-EGFP和Bac-inrep,并将二者的P3代病毒共同感染昆虫细胞Spodoptera frugiperda(Sf9),抽提小分子量DNA,获得AAV-ITR-EGFP基因表达微载体,2×107的Sf9细胞抽提可以得到100μg AAV-ITR-EGFP基因表达微载体,核酸电泳显示AAV-ITR-EGFP基因表达微载体主要以单体和二聚体的形式存在。将AAV-ITR-EGFP基因表达微载体通过polyethylenimine(PEI)转染HEK 293T细胞,24 h后荧光显微镜观察有EGFP表达,48 h后达到高峰,转化效率达到65%。     

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.     

Estrogen receptor beta yield from baculovirus lytic infection is higher than from stably transformed Sf21 cells

The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERβ than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERβ decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERβ expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERβ production than stable-transformation of Sf21 cells.     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.     

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ～150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme‐Nitric Oxide Oxygen (H‐NOX), Per‐ARNT‐Sim (PAS), coiled‐coil and cyclase domains. Here, we present the crystal structure of the α₁ PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β₁ H‐NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.     

Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae in Sf21 insect cells

The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.     

Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. We expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [3H]-azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents (e.g., daunomycin greater than verapamil greater than vinblastine approximately vincristine) compete with the [3H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.