Construction Of Recombinant Baculovirus Research Articles

Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity.

Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses.

Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases.

Development of a vectored vaccine against Hepatitis E virus

Hepatitis E virus is a non-enveloped ssRNA virus [1] that causes human acute hepatitis through primarily fecal and oral transmission [2]. Currently, no commercial hepatitis E (HEV) vaccine is available. In the absence of an appropriate cell culture system for HEV propagation, HEV pseudocapsids (ORF2 protein) have been produced either in Escherichia coli or in insect cells and they have been shown to protect monkeys against virus challenge and to be effective in the prevention of natural HEV infection of humans.In this work, we propose to develop a novel candidate vaccine against hepatitis E infection using adeno-associated virus (AAV) as a vector expressing the gene of the truncated capsid protein of HEV (aa 112–aa 660). rAAV will be produced in Sf9 cells using the baculovirus expression vector system.For this purpose, construction of recombinant baculoviruses was performed and viral stocks of BacRep, BacCap for serotypes 2, 5 and 6 were prepared in Sf9 cells. The recombinant baculovirus coding for the truncated capsid protein of HEV (BacITRHEVORF2) was also constructed, the virus titer was equal to 5.41×109PFU/mL, at the third passage. Transduction of HEK 293 EBNA cells with rAAV was carried out; the production of HEVORF2 was confirmed by Western blot. Optimization of rAAV production in Sf9 cells is currently ongoing.

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

Construcción de bacmids recombinantes con genes inmunogénicos del virus de la fiebre aftosa usando el sistema Bac-to-Bac

El virus de la fiebre aftosa pertenece a la familia de los Picornavirus y afecta mayormente a animales bovinos, ovinos, porcinos y caprinos. Se divide en 7 serotipos: O, A, C, Sat1, Sat2, Sat3 y Asia1. En Latinoamérica, los serotipos más prevalentes son el O y A, en Ecuador, el serotipo de mayor incidencia es el O. La vacuna que se utiliza actualmente es una vacuna convencional de virus inactivado que posee algunas desventajas como una respuesta inmune limitada, la necesidad de una cadena de frío para mantener a la vacuna viable y el requerimiento de grandes cantidades de virus para fabricarla. Una alternativa para superar estos problemas y en general los problemas de las vacunas convencionales, es la creación de subunidades de vacunas por medio de un sistema de expresión de proteínas recombinantes, dónde se utiliza sólo las proteínas inmunogénicas del patógeno. Por lo tanto, este proyecto se centró en la aplicación del sistema Bac-to-Bac para la construcción de baculovirus recombinantes con genes de la cápside, altamente inmunogénicos, del virus de la fiebre aftosa como son VP1, P1-2A y 3C del serotipo O. Se logró obtener baculovirus recombinantes con el constructo VP1 y éstos, posteriormente, serán transfectados en células de insectos para la expresión y análisis de VP1 como un posible candidato de subunidad de vacuna.

Costs and Consequences of Cellular Compartmentalization and Substrate Competition among Human Enzymes Involved in Androgen and Estrogen Synthesis

The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.

Construction and immunogenicity of a recombinant pseudotype baculovirus expressing the glycoprotein of rabies virus in mice

A pseudotype baculovirus with the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope was used as a vector for the construction of recombinant baculovirus expressing the G protein of rabies virus (RABV) under the cytomegalovirus (CMV) promoter. The generated recombinant baculovirus (BV-G) efficiently expressed the RABV G proteins in mammalian cells. Intramuscular vaccination with BV-G (10(9) PFU/mouse) induced the production of RABV G-specific neutralizing antibodies and strong T cell responses in mice. Our data clearly indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccine against RABV infection.

Large-scale production of functional recombinant CAR in a baculovirus- insect cell system

Coxsackievirus group B and adenovirus receptor (CAR) is a major receptor for the adenovirus groups that has drawn overall attention over the past decade. Although this protein could potentially be used as an agent for the blocking of adenovirus infection, large-scale production of highly purified human CAR in eukaryotic expression system has not been reported. In the present study, we showed the construction of recombinant baculovirus highly-expressing the extracellular domain of human coxsackievirus-adenovirus receptor (exCAR) in High Five insect cells. The recombinant exCAR was recovered from the cell culture medium as a secreted soluble protein and purified by Ni-NTA affinity chromatography. The final yield of recombinant exCAR was about 8-10 mg/l of supernatant with the purity of 96.3%. Binding activity assay showed that the recombinant exCAR exhibited an intact ability of binding to the knob domain of the adenovirus type 5 fiber protein (Ad fiber knob) displayed by T7 phage. These results showed that the recombinant human exCAR produced in insect cells and purified by Ni-NTA chromatography retained its ability to bind to the Ad fiber knob and could potentially be used in therapy of adenovirus infection.

Characterisation of a GII-4 norovirus variant-specific surface-exposed site involved in antibody binding

BackgroundThe human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion. The noroviruses are classified on the basis of nucleotide sequence diversity in the VP1-encoding ORF2 gene, which divides the majority of human noroviruses into two genogroups (GI and GII). GII-4 noroviruses are the major aetiological agent of outbreaks of gastroenteritis around the world. During a winter season the diversity among the GII-4 noroviruses has been shown to fluctuate, driving the appearance of new virus variants in the population. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B) in the hypervariable P2 domain. We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants.ResultsIn this paper, we describe the construction of recombinant baculoviruses to express VLPs representing one pre-epidemic and one epidemic variant of GII-4 noroviruses, and the production of monoclonal antibodies against them. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding.ConclusionAs predicted by our earlier study, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that are antibody escape mutants.

Filovirus‐Like Particles Produced in Insect Cells: Immunogenicity and Protection in Rodents

Virus-like particles (VLPs) of Ebola virus (EBOV) and Marburg virus (MARV) produced in human 293T embryonic kidney cells have been shown to be effective vaccines against filoviral infection. In this study, we explored alternative strategies for production of filovirus-like particle-based vaccines, to accelerate the development process. The goal of this work was to increase the yield of VLPs, while retaining their immunogenic properties. Ebola and Marburg VLPs (eVLPs and mVLPs, respectively) were generated by use of recombinant baculovirus constructs expressing glycoprotein, VP40 matrix protein, and nucleoprotein from coinfected insect cells. The baculovirus-derived eVLPs and mVLPs were characterized biochemically, and then the immune responses produced by the eVLPs in insect cells were studied further. The baculovirus-derived eVLPs elicited maturation of human myeloid dendritic cells (DCs), indicating their immunogenic properties. Mice vaccinated with insect cell-derived eVLPs generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell-derived eVLPs and were protected from EBOV challenge in a dose-dependent manner. Together, these data suggest that filovirus-like particles produced by baculovirus expression systems, which are amenable to large-scale production, are highly immunogenic and are suitable as safe and effective vaccines for the prevention of filoviral infection.

Directional and Direct Cloning Strategy for High-Throughput Generation of Recombinant Baculoviruses

The baculovirus expression vector system (BEVS) has become one of the most widely used systems for routine protein expression. We have developed an improved strategy to clone foreign genes directionally and directly into the baculovirus genome vector via a one-step procedure to generate recombinant viruses in a week. In this work, we constructed a host strain Escherichia coli DH10BacHB1.1, which contains the modified baculovirus shuttle genome vector pHBMBacmid1.1 for the cloning vector. The treated PCR products of foreign genes were ligated with the Bsu36I-digested vector. Then Spodoptera frugiperda (Sf9) cells were transfected directly with the ligation mixture. Using this method, the DsRed fluorescence protein and mannanase genes have been cloned in the baculovirus genome and expressed in the Sf9 cells. This strategy not only provides a means for high-throughput construction of recombinant baculoviruses, but also offers an idea of constructing other large plasmids and DNA virus-based expression vectors.

Variation in Ligand Binding Specificities of a Novel Class of Poxvirus-encoded Tumor Necrosis Factor-binding Protein

The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but distinct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors from Tanapox virus, Yaba monkey tumor virus (YMTV) and a closely related version from Swinepox virus revealed dramatically differential TNF binding specificities for different mammalian species. The Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of these TNF-binding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhibited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD, 43 pm) compared with monkey (KD, 120 pm) whereas for YMTV-2L, the affinities were reversed (human TNF KD, 440 pm; monkey TNF KD, 230 pm). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as determined using human TNF mutants. We conclude that these poxvirus TNF-binding proteins represent a new class of TNF inhibitors and are distinct from the viral TNF receptor homologues characterized to date.

Suppression of HCV RNA replication by baculovirus-mediated shRNA expression

We describe a HCV core-short-hairpin RNA (shRNA) expressed from a human U6 Pol II promoter, which synergistically enhanced the inhibition of HCV replication in HCV replicon. We also describe the construction of recombinant baculovirus expressing shRNAs of the HCV core gene under the control of the U6 promoter. The anti-HCV shRNAs may provide a practical basis for applying shRNA-based gene therapy to the treatment of HCV.

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.

Characterization of novel benzodiazepine ligands in Spodotera frugiperda (Sf-9) insect cells

The goals of the present work were to characterize the binding profile of nine benzodiazepine ligands in Spodotera frugiperda (Sf-9) insect cells expressing specific gamma aminobutyric acid A (GABA A) receptor subunit combinations and compare the affinities to those for the receptors in the rat cerebellum. Three recombinant baculovirus constructs, each harboring a different GABA A receptor subunit, were introduced into insect cells by simultaneous infection. Saturation and competition binding assays were carried out in membranes from Sf-9 cells infected with either α 1 β 2 γ 2 or α 6 β 2 γ 2 subunit combinations. The affinities of the ligands to the α 1 β 2 γ 2 or α 6 β 2 γ 2 receptors expressed in Sf-9 cells were similar to the affinities previously determined for the α 1 or α 6 subunit-containing GABA A receptors in the rat cerebellum, respectively, thus confirming the previously assigned receptor types in the cerebellum.

30] Baculovirus-mediated expression of neurotransmitter transporters

Publisher Summary This chapter describes baculovirus-mediated expression of neurotransmitter transporters. Na + /Cl − dependent neurotransmitter transporters constitute a small family of integral membrane proteins that couple the uptake of small molecules to the movement of ions down their concentration gradients. The determination of the three-dimensional structure of at least one member of the neurotransmitter transporter family is essential to understand how they transport molecules into the cell and how inhibitors work. However, the majority of these transporters are normally expressed at low levels and only the γ-aminobutyric acid (GABA) transporter (GAT), serotonin transporter (SERT), and glycine transporter are purified from natural sources. The structural characteristics of the neurotransmitter transporters make them among the most poorly overexpressed membrane proteins, regardless of the expression system used. This chapter explains the construction of recombinant baculovirus expressing neurotransmitter transporters and their subsequent characterization.

Expression of Functional Melanocortin 1 Receptors in Insect Cells

We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4, D-Phe7]α-MSH. Both saturation and competition analysis, using α-, β-, and γ1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells.

Cassette baculovirus vectors for the production of chimeric, humanized, or human antibodies in insect cells.

Plasmid cassette-transfer vectors pBHuC chi and pBHuC gamma l have been designed which enable the construction of recombinant baculoviruses directing the co-expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti-human CD29 monoclonal antibody (Mu-K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch-K20-sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype.

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.