Insect Cell Extracts Research Articles

A Plumieridine-Rich Fraction From Allamanda polyantha Inhibits Chitinolytic Activity and Exhibits Antifungal Properties Against Cryptococcus neoformans.

Cryptococcosis is a fungal infection caused mainly by the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii. The infection initiates with the inhalation of propagules that are then deposited in the lungs. If not properly treated, cryptococci cells can disseminate and reach the central nervous system. The current recommended treatment for cryptococcosis employs a three-stage regimen, with the administration of amphotericin B, flucytosine and fluconazole. Although effective, these drugs are often unavailable worldwide, can lead to resistance development, and may display toxic effects on the patients. Thus, new drugs for cryptococcosis treatment are needed. Recently, an iridoid named plumieridine was found in Allamanda polyantha seed extract; it exhibited antifungal activity against C. neoformans with a MIC of 250 μg/mL. To address the mode of action of plumieridine, several in silico and in vitro experiments were performed. Through a ligand-based a virtual screening approach, chitinases were identified as potential targets. Confirmatory in vitro assays showed that C. neoformans cell-free supernatant incubated with plumieridine displayed reduced chitinase activity, while chitinolytic activity was not inhibited in the insoluble cell fraction. Additionally, confocal microscopy revealed changes in the distribution of chitooligomers in the cryptococcal cell wall, from a polarized to a diffuse cell pattern state. Remarkably, further assays have shown that plumieridine can also inhibit the chitinolytic activity from the supernatant and cell-free extracts of bacteria, insect and mouse-derived macrophage cells (J774.A1). Together, our results suggest that plumieridine can be a broad-spectrum chitinase inhibitor.

Identification of the Epstein Barr Virus portal

Little is known about Epstein Barr Virus (EBV) proteins that participate in viral DNA cleavage and packaging. Genes encoding potential terminase subunit and portal protein homologs include BGRF1/BDRF1, BALF3, BFRF1A and BBRF1 respectively. EBV mutants with deletions in one or more of these genes were impaired for DNA packaging (Pavlova et al., 2013). In the current study, BBRF1 oligomers were purified from recombinant baculovirus infected insect cell extracts. Transmission electron microscopy revealed that purified EBV portals retained features typically found in other portals including a central channel with clip, stem and wing/crown domains. Although compounds have been identified that target DNA encapsidation in human cytomegalovirus, herpes simplex viruses and varicella-zoster virus, the identification of new EBV targets has lagged significantly. Characterization of the EBV portal will direct studies aimed at developing potential small molecular inhibitors of the EBV encapsidation process.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individualproteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5'→3' Xrn Exoribonuclease Activity.

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the ‘core’ nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this ‘core’ sequence resides a conserved “coremin” motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing “coremin” at its 5′ end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5′-to-3′ exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 ‘core’ sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.

Biochemical analysis of NSs from different tospoviruses

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.

PDE9 INHIBITION IMPROVES COGNITION IN MICE BY A MECHANISM DIFFERENT FROM THE ACETYLCHOLINE ESTERASE INHIBITOR DONEPEZIL

Phosphodiesterases (PDEs) in the brain have shown to play an important role in synaptic plasticity and cognitive function. Within this target class is PDE9, which specifically regulates the second messenger cGMP in neurons related to NMDA receptor signaling and is expressed in cognition relevant regions of the brain. Thus, PDE9 inhibitors are hypothesized to improve cognitive function via increasing NMDA receptor related cGMP signalling pathway to strengthen synaptic plasticity. This study characterizes the effect of a PDE9 inhibitor (compound A) on reversal of cognitively impaired mice in the T-maze task. In addition, brain cGMP levels were measured in these mice and correlated with memory performance in this task, and furthermore, compared with the effects of the acetylcholine esterase inhibitor donepezil. The molecular potency of compound A for PDE9 was determined using cytosolic extracts of SF9 insect cells over-expressing full-length human enzyme employing SPA technology. Regarding cognition, compound A was tested for reversal of MK-801 induced memory impairment in the mouse T-maze spontaneous alternation task after intraperitoneal administration. Subsequently, cGMP levels in mouse prefrontal cortex, hippocampus and striatum were measured by using enzyme immunoassay technique. The IC50 value of compound A on PDE9 was determined to be 88 nM. Regarding cognition, the PDE9 inhibitor could improve MK-801 induced memory deficits in the mouse T-maze task which was positively correlated with cGMP increase in prefrontal cortex, hippocampus and striatum, brain regions relevant for performance in this task. Donepezil also led to an improvement of memory deficits in the T-maze task, but this was not correlated with cGMP increase in brain. The study shows that compound A is a potent PDE9 inhibitor, and - corroborating previous reports on memory enhancing efficacy of other PDE9 inhibitors in rodents - it shows cognition enhancing efficacy in the mouse T-maze task. In contrast to donepezil, improvement of memory performance by PDE9 inhibition was correlated with a cGMP increase in cognitively relevant brain regions. Therefore, these data further demonstrate that PDE9 inhibition may be a potential approach to pharmacologically improve cognition in patients with Alzheimer's disease by a different mechanism compared to donepezil.

Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications.

During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments.

Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling.

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

CRY4AA AND CRY4BA FROM BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS EXPRESSED IN INSECT CELLS BY RECOMBINANT BACULOVIRUSES ARE TOXIC TO AEDES AEGYPTI LARVAE

Bacillus thuringiensis subsp. israelensis (Bti) is highly toxic to mosquito larvae. Most of its toxicity relies on δ-endotoxins (Cry and Cyt) crystalline inclusions produced at the sporulation phase of growth. Obtention of individual Bti mosquitocidal toxins is crucial for the understanding of Bti´s potential activity against dipteran larvae. A Cry toxin expression system based on baculovirus was developed to produce two of the Cry toxins comprised in the crystals of Bti. Th e cry4Aa and cry4Ba genes from two Brazilian strains of Bacillus thuringiensis were amplified by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis of the cry4Aa and cry4Ba genes showed high identity to previous known cry genes. Both cry4Aa and cry4Ba genes were further cloned into transfer vectors for construction of recombinant baculoviruses. After isolation of the recombinant viruses, they were used to infect insect cells (BTITN5B1-4) that were analyzed by light microscopy at different times post infection. Putative crystals consisting of either Cry4Aa or Cry4Ba were observed in the cytoplasm of infected insect cells. RT-PCR was performed with mRNA from insect cell extracts (72 h.p.i.) in order to confirm the presence of the genes specific transcripts. Recombinant virus-infected insect extracts (120 h.p.i.) were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) showing the presence of polypeptide bands of around 128 and 130 kDa, corresponding, respectively to the sizes of the proteins Cry4Aa and Cry4Ba. Bioassays with virus-infected insect extracts were shown to be toxic to second instar Aedes aegypti larvae, confirming the usefulness of this expression system for the study of Cry proteins. DOI: http://dx.doi.org/10.17525/vrr.v18i1-2.70

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

Antihypertensive effect of insect cells: In vitro and in vivo evaluation

In this study, we investigated the in vitro ACE inhibitory and in vivo antihypertensive effect of insect cell extracts. The IC 50 of three insect cell lines from different type and insect species origin: S2 (embryo, Drosophila melanogaster), Sf21 (ovary, Spodoptera frugiperda) and Bm5 (ovary, Bombyx mori), were evaluated. Most interesting results were that the IC 50 values ranged between 0.4 and 0.9 mg/ml, and that an extra hydrolysis with gastrointestinal enzymes did not increase the ACE inhibitory activity conspicuously. Finally, a single oral administration with a gavage of 150 mg cell extract/kg BW to spontaneous hypertensive rats (SHR) significantly decreased ( p < 0.05) their systolic blood pressure (SBP) with 5–6% (9–12 mm Hg) compared to the controls at 6 h post-administration. Here the undigested and digested insect S2 cell extracts were equal in activity to lower the SBP. To the best of our knowledge, this is the first report of in vivo antihypertensive activity of insect cell extracts and this without an extra digestion requirement.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

Monolayer purification: A rapid method for isolating protein complexes for single-particle electron microscopy

Visualizing macromolecular complexes by single-particle electron microscopy (EM) entails stringent biochemical purification, specimen preparation, low-dose imaging, and 3D image reconstruction. Here, we introduce the "monolayer purification" method, which employs nickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simultaneously purifying His-tagged complexes directly from cell lysates while producing specimens suitable for single-particle EM. The method was established by using monolayers containing Ni-NTA lipid to specifically adsorb His-tagged transferrin-transferrin receptor (Tf-TfR) complexes from insect and mammalian cell extracts. The specificity and sensitivity of the method could be improved by adding imidazole to the extracts. The monolayer-purified Tf-TfR samples could be vitrified and used to calculate a 3D reconstruction of the complex. Monolayer purification was then used to rapidly isolate ribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowed calculation of a cryo-EM 3D reconstruction of the Escherichia coli 50S ribosomal subunit.

Oral immunization with different assembly forms of the HPV 16 major capsid protein L1 induces neutralizing antibodies and cytotoxic T-lymphocytes

Human papillomaviruses have been recognized as the causative agent of anogenital cancer. In 2006, a commercial vaccine based on virus-like particles composed of the L1 major capsid protein of the papillomaviruses has been available. This vaccine induces virus-neutralizing antibody responses upon parenteral injection. Here we investigated the oral immunogenicity of different assembly forms of HPV 16 L1, that is: T7-VLPs, T1 particles and capsomeres. Our results show that all three assembly forms induce humoral and cellular immune responses after oral vaccination of mice. The anti-L1 antibodies were conformation-specific and showed neutralizing activity in a pseudovirion-based assay. We also investigated if adjuvants have an influence on the oral immunogenicity of the different L1 forms. For saponins we observed a significant toxicity if applied orally. Co-administration of either CpG DNA or Escherichia coli heat-labile enterotoxin LT(R192G) had no apparent enhancing effect on the production of anti-L1 antibodies. More pronounced was the effect of CpG administration on the long-term immunity as we observed a significantly stronger recall response 244 days after the first vaccination. Compared to capsomeres, VLPs induced stronger humoral immune responses while the CTL responses were induced at comparable levels. Finally, we were also able to induce neutralizing antibodies and L1-specific cytotoxic T-lymphocytes after oral administration of crude extracts of L1-expressing insect cells. In conclusion, all three assembly forms of the L1 protein are immunogenic when administered orally.

A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL(50) of 19.49 ng/ml for 2(nd) instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Human methionine synthase reductase is a molecular chaperone for human methionine synthase.

Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K(act) value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37 degrees C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS.

CGMP-dependent Protein Kinase Phosphorylates p21-activated Kinase (Pak) 1, Inhibiting Pak/Nck Binding and Stimulating Pak/Vasodilator-stimulated Phosphoprotein Association

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.

Cell‐Free Protein Synthesis System Prepared from Insect Cells by Freeze‐Thawing

We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method.

Increasing the ease and speed of eukaryotic protein expression: a new cell-free in vitro translation system based on Sf insect cell extracts

Background For researchers looking for fast access to their protein of interest, cell-free expression systems are an attractive option, as they do not require specialized equipment, are open systems, and offer fast screening of expression constructs for expression efficiency, yield, solubility and other criteria (e.g. requirement of cofactors). A broad range of eukaryotic proteins require posttranslational modifications such as phosphorylation, glycosylation, or signal peptide cleavage to display full functional activity.