Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells

Rho-kinase II (ROCK-II) is a serine/threonine kinase that is involved in regulation of smooth muscle contraction and has been shown to contribute to the early stages of axon formation in neurons and the regulation of the neuronal cytoskeleton. Much of what is known about Rho-kinase function comes from cell-biological studies, whereas a paucity of biochemical characterization exists for the enzyme. In an effort to characterize ROCK-II biochemically we have cloned a truncated form of human ROCK-II comprising amino acids 1-543 and overexpressed it in Sf-21 cells. Utilizing the Sf-21/baculovirus expression system we isolated milligram quantities of ROCK-II (1-543) and purified the enzyme to near homogeneity. Optimal expression conditions revealed that infection of Sf-21 cells at a multiplicity of infection of 10 for 72h yielded maximal protein expression. Expression of ROCK-II (1-543) as an N-terminal Flag fusion protein allowed a single-step purification yielding greater than 90% homogeneous protein as assessed by SDS-PAGE. Enzyme activity was linear over a range of enzyme concentrations and times. Capture of phosphorylated, biotinylated peptides on streptavidin membrane allowed assessment of peptide substrate preference and measurement of steady-state rate constants. The data indicated that an 11-mer peptide containing Ser235/Ser236 of the S6 ribosomal protein and a 12-mer peptide containing Thr508 of LIM kinase were preferred substrates for ROCK-II (1-543). Finally, staurosporine had an IC(50) value 215-fold more potent than that of the ROCK inhibitor Y-27632. Collectively these data lay the foundation for the beginning of a biochemical characterization for this enzyme and provide methodology for more detailed biochemical, biophysical, and kinetic analysis.     

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%.     

Mathematical characterization of insect cell (Sf-9) death in suspended culture

The effect of baculovirus infection on cell death in suspended cultures was characterized based on work by Wu et al. (1993) Biotech. Bioeng. 41: 104–110 and Wu et al. (1994) Biotechnol. Prog. 10: 55–59. The post infection time can be separated into a constant viability phase characterized by a time delay, td, and a rapid death phase, which is characterized by a specific death rate constant, k. Results indicated that the characteristic time delay decreased with increased multiplicity on infection (MOI). Further, there was only a weak correlation between specific death rate and MOI, for the range of MOI tested. Cell infection and death rates were consistent with a more evenly distributed infection process likely found in suspension cultures.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni²⁺-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 °C and −20 °C, but were well preserved at −80 °C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.     

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.     

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.     

Expression, purification, characterization and crystallization of a recombinant human cytosolic beta-glucosidase produced in insect cells

Human cytosolic beta-glucosidase is a monomeric enzyme that hydrolyzes various beta-d-glycosides and its real physiological role remains unclear. Here, we describe the production of this enzyme in Sf9 cells with a N-terminal 6x His tag. The production yield of the recombinant protein was in the 10 to 30 mg/l range. The protein was purified to homogeneity using two chromatographic steps, taking advantage of the 6x His tag in the first step, then using the physical and chemical properties of the protein for ionic exchange. Gel filtration analysis revealed that the protein is monomeric as expected. The kinetic parameters for 4-methylumbelliferyl beta-L-glucopyranoside, VM and KM, were measured (KM=32 microM and VM=157 micromol/h/mg at pH 7.0) and found similar to those reported for either the natural isolated enzyme or the recombinant protein expressed in COS7 cells (KM of 60-70 microM and 40 microM, respectively). Protein crystals were obtained and are now under structural investigations. In summary, we set up a heterologous expression system in Sf9 insect cells allowing the expression and production of large amounts of a pure active human protein, suitable for crystallographic studies.     

Substrate (gelatin) gel electrophoretic method for analysis of protease activity in insect (Sf-9) cells

A method for detection, quantification, and characterization of protease activities present in the insect cell/baculovirus protein expression system was demonstrated. Proteases were found of 39, 44, and 52 kDa molecular weight. Enzymatic inhibitors (antipain, chymostatin, EDTA, pepstatin A), cofactors (e.g., ATP, Mg 2+ , Ca 2+ ), pH, and substrates (gelatin, b-galactosidase, bovine serum albumin) were varied to improve and characterize the activities.     

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli

We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli. The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF. The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography. The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture. Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF.     

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.     

Functional characterization and purification of the secretin receptor expressed in baculovirus-infected insect cells

Structural insights into Class II G protein-coupled receptors have been limited by the absence of a plentiful and highly enrichable source such as rhodopsin in the Class I family. With structural differences predicted to exist between these families, and with the key importance of an intact, disulfide-bonded amino-terminal domain for the Class II receptors, an overproduction and purification scheme is critically important. In this work, we have established and characterized a baculoviral expression and purification system for the secretin receptor. Hemagglutinin epitope-tagged wild-type rat secretin receptor construct was expressed using the recombinant baculovirus/Sf9 insect cell-based system, achieving a level of expression substantially higher than that previously achieved in Chinese hamster ovary (CHO-SecR) cells. Receptor expressed in Sf9 cells had similar affinity for secretin (Ki=1.4+/-0.2 nM) and similar potency to stimulate intracellular cAMP in response to this hormone (EC50=194+/-45 pM) as did wild-type receptor expressed in CHO cells. Receptors from Sf9 cells were also affinity labeled saturably and specifically by a photolabile secretin analogue. The receptors were purified to homogeneity by solubilization with sodium deoxycholate, selective ammonium sulfate precipitation, gel filtration and immunoaffinity purification. This expression system should facilitate the structural characterization of this receptor and its important amino-terminal domain.     

Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.     

Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells

Testican-2 is a member of the testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was approximately 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit cathepsins L, B, K and S. We identified the C1q subcomponent of complement component C1 as a potential interacting partner of testican-2. The C1q subcomponent is a recognition molecule which acts in concert with other C1 subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes.     

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.     

Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.     

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.     

Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells

The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.