Enzyme Activity Expression Research Articles

Regulating Strategies for Producing Carbohydrate Active Enzymes by Filamentous Fungal Cell Factories.

Filamentous fungi are important eukaryotic organisms crucial in substrate degradation and carbon cycle on the earth and have been harnessed as cell factories for the production of proteins and other high value-added products in recent decades. As cell factories, filamentous fungi play a crucial role in industrial protein production as both native hosts and heterologous hosts. In this review, the regulation strategies of carbohydrate active enzyme expression at both transcription level and protein level are introduced, and the transcription regulations are highlighted with induction mechanism, signaling pathway, and promoter and transcription factor regulation. Afterward, the regulation strategies in protein level including suitable posttranslational modification, protein secretion enhancement, and protease reduction are also presented. Finally, the challenges and perspectives in this field are discussed. In this way, a comprehensive knowledge regarding carbohydrate active enzyme production regulation at both transcriptional and protein levels is provided with the particular goal of aiding in the practical application of filamentous fungi for industrial protein production.

Iterative Methylations Resulting in the Biosynthesis of the t-Butyl Group Catalyzed by a B12-Dependent Radical SAM Enzyme in Cystobactamid Biosynthesis.

B12-dependent radical SAM enzymes that can perform methylations on sp3 carbon centers are important for functional diversity and regulation of biological activity in several nonribosomal peptides. Detailed studies on these enzymes are hindered by the complexity of the substrates and low levels of expression of active enzymes. CysS can catalyze iterative methylations of a methoxybenzene moiety during the biosynthesis of the cystobactamids. Here, we describe the overexpression, purification, substrate identification, and mechanism of this enzyme.

A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli

Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL−1 cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.

Grape Processing by High Hydrostatic Pressure: Effect on Use of Non-Saccharomyces in Must Fermentation

Vitis vinifera (variety Tempranillo) grapes were pressurized at 400 MPa by high hydrostatic pressure for 10 min and the effect on wild microbial populations, phenol extraction and wine composition was monitored. After treatment, the grapes were inoculated and fermented with Saccharomyces cerevisiae and several non-Saccharomyces yeasts: Schizosaccharomyces pombe, Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans. S. pombe was used as sole fermentative yeast, but T. delbrueckii, M. pulcherrima and L. thermotolerans were used in sequential cultures with S. cerevisiae to completely ferment the sugars. The HHP treatment reduces strongly or eliminates wild microorganisms, especially yeasts, facilitating the growth and development of non-Saccharomyces yeasts. Thus, it helps to get either a better expression of enzymatic activities or metabolites production of non-Saccharomyces affecting wine quality.

Nuclease Activity Associated with Secreting Granules by Lymphocytes in Patients with Bronchial Asthma

Background: We know, through recent studies, the existence of some morpho-biochemical peculiarities in the process of type 1 programmed death of patients’ lymphocytes suffering from bronchial asthma, but little convincing data exist on the activity of enzymes involved in this physiological process. Therefore, the aim of our research was to study the enzymatic activity of secreting granules of patients’ lymphocytes with bronchial asthma, according to the degree of severity. Method: The study was based on the role of granular extracts in the process of programmed death isolated lymphocytes from peripheral blood of relatively healthy individuals and asthmatic patients with different severity. The immunological characteristics of lymphocytes was done with the radial immune-diffusion method and ELISA test but the method of agarose gel electrophoresis help us to detect the catalytic activity of protein extracts of secreting granules of lymphocytes. Results: The results obtained showed that lymphocytes from asthmatic patients with severe severity are characterized by a decrease in cytotoxic T lymphocytes content balanced by an increase in T-Helper lymphocytes. We also noticed the enzymatic activity at all the groups studied but this activity was relatively high in asthmatics with severe severity. Furthermore, the study of the cationic dependence has allowed to establish an increase in enzymatic activity in all the groups studied after incubation of DNA in a medium containing Ca2+ with a pH of 7.5 unlike ions Mn2+ which seem to reduce the enzymatic activity. The expression of enzymatic activity in the presence of zinc allows us to suggest the presence of DNase acid in granules, which activity is not necessarily associated with divalent metal ions. Conclusion: Based on the above results, one might conclude that the secreting granules have a high enzymatic activity but with a strong cationic dependence. This not only allows a better understanding of the morphological changes observed during the course of apoptosis in lymphocytes of patients but also brings more to the knowledge of the enzymatic influence in the process of type 1 programmed death.

Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity.

CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.

Effects of temperature and incubation time on the in vitro expression of proteases, phospholipases, lipases and DNases by different species of Trichosporon.

Fungi produce a broad spectrum of enzymes capable of degrading different substrates in nature. When the substrate is the tissue of a vertebrate host, these enzymes acts as a fungal virulence factor that increases the pathogenicity of the fungus. Trichosporon yeasts are emerging pathogens that infect immunocompromised patients. Little is known about the virulence characteristics of these fungi. The aim of this research was to characterize the behavior of protease, phospholipase, lipase and DNase production in different species of Trichosporon, with a focus on the influence of incubation temperature on the expression of these enzymes. Classical methodologies were used in all of the experiments, and the results were statistically analyzed. The proportions of the samples that produced each type of enzyme were as follows: lipases (95.5%), phospholipases (56.8%), proteases (50,0%) and DNases (38.6%). The incubation temperature was an important factor in the expression of enzymatic activity, and it influenced the incubation period of each species. Although these data concerning the enzymatic activity expressed by isolates of Trichosporon are valuable, further research is warranted to completely characterize this new pathogen, as well as in vivo studies to determine the roles of these enzymes in the pathogenesis of trichosporonosis.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis

BackgroundYedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.ResultsIn this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.ConclusionOur study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

RECOMBINANT HORSERADISH PEROXIDASE FOR ANALYTICAL APPLICATIONS

86 Horseradish peroxidase isoenzyme C (HRP, EC 1.11.1.7), a hemeand Ca-contai ning glycoprotein, is a member of the superfa mily of plant peroxidases [1–3] that are able to utilize hydrogen peroxide to catalyze the one electron oxidation of a wide variety of organic and inorganic substrates. Whereas plant pe roxidases find interest for applications in va rious biotechnological processes, like bleaching and degradation of organic compounds (e.g. phenols and lignin), HRP specifically is used in analytical biochemistry and biotechnology as a marker enzyme for antibodies, DNA and low molecular mass analytes. Broad substrate specificity and high catalytic activity and stability determined the world-wide application of HRP in bioand immunosensors, chemiluminescent, fluorescent and electrochemical detection systems, DNA microarrays and biochips with HRP-based colori metric detection [4–6, 14, 56]. Progress made in peroxidase gene hetero logous expression opened up the prospect to study structure-function relationships by means of genetic engineering. The baculovirus expression system allowed production of recombinant HRP in soluble, active and glycosylated form [7], however, this system is laborious and not as wide spread as expression in E. coli. Expression of HRP in yeast indicated only very low expression of active enzyme [8]. Up to date for the production of wild-type as well as mutant recombinant HRP the E. coli expression system is mostly used [9, 10]. Recombinant HRP forms inclusion bodies containing only traces of heme if expressed in bacterial cytoplasm. Multistep refolding and reactivation of recombinant apo-peroxidase with the prosthetic heme group is complex as the protein contains four disulfide bonds and in addition must bind two Ca-ions per molecule. Moreover, the plant-derived enzyme contains 18% carbohydrates via 8 glycosylation sites. Crystal structure of recombinant HRP has been solved, revealing the presence of two domains formed by a total of ten α-helices [11]. Among of the factors leading to the formation of inclusion bodies upon expression of UDK 543.645.6:665.123

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2+ dependent and catalyzes a similar SN2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate=20±4μM, KMg2+=294±22μM, KL-gln=11±1mM, and kcat=2.2±0.2s−1 for a random kinetic mechanism. PhzE can use NH4+ as an alternative nucleophile, while Co2+ and Mn2+ are alternative divalent metals.

C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli

Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases ( Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc- l-Ala- l-Ala- l-Pro- l-Phe- p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in K M = 0.23 ± 0.01 mM and k cat = 167.5 ± 3.6 s −1. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.

Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into alpha-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5alpha proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.

Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris

Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119–138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

Pectin methylesterase, polyphenol oxidase and physicochemical properties of typical long‐storage cherry tomatoes cultivated under water stress regime

AbstractBACKGROUND: The selection of cherry tomato genotypes with extended shelf‐life and resistant to water stress is an important aspect of Sicilian agriculture. The aim of the present study was to evaluate the effects of watering regime on the expression of enzymatic activities (pectin methylesterase (PME, EC 3.1.1.11) and polyphenol oxidase (PPO, EC 1.14.18.1)) and the physicochemical properties of typical long‐storage cherry tomatoes in order to propose a solution strategy suitable for Sicilian cultivation.RESULTS: PPO activity seemed to be related to the watering regime employed, showing a maximum under normal watering conditions. PME activity also decreased greatly with increasing water stress (loss of activity ranging from 22.31 to 82.01%). Water stress induced an increase in titratable acidity (up to 68.7% in cultivar ‘Giallo piccolo a punta’) and, above all, in dry matter and total soluble solids (increases ranging from 24.08 to 91.68% and from 5.93 to 40.90% respectively). Possible solution strategies for cultivation of tomato genotypes with extended shelf‐life are (a) selecting the local type that presents the lowest levels of PME and PPO (‘Pizzutello’) or (b) modifying the watering regime in order to induce commercial cultivars employed in intensive cultivation (e.g. ‘Naomi’) to better protect themselves from degradative enzymatic activities.CONCLUSION: It appears that the adoption of high watering levels, leading to a more marked expression of degradative enzymatic activities, could result in cherry tomatoes with a high susceptibility to undesirable browning and softening reactions. Copyright © 2007 Society of Chemical Industry

Expression of active enzymes from an inducible tomato-mosaic-virus-based vector in cultured transgenic tobacco BY-2 cells

Previously, we reported on an inducible viral vector system for foreign protein production in cultured plant cells, in which the transcription of the recombinant viral vector RNA encoding a foreign protein is controlled by an estradiol-inducible promoter. In this study, we used the inducible virus vector system to test the efficiency of a modified tomato mosaic virus (ToMV) encoding the movement protein. The virus inducibly produced a foreign jellyfish green fluorescent protein encoded in the virus in transgenic tobacco BY-2 suspension-cultured cells as efficiently as modified ToMV without the movement protein. We then produced transgenic BY-2 cell lines ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS, which encoded modified ToMV with Escherichia coli dihydrofolate reductase (DHFR) and β-glucuronidase (GUS), respectively. After estradiol was added to the cell culture, DHFR and GUS activity was detected in the ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS cells, respectively. In contrast, no DHFR or GUS activity was detected in untreated transgenic lines. Three days after induction, DHFR accumulation accounted for up to 15% of the total soluble proteins extracted from the cells, indicating that an inducible viral vector is an effective option for efficiently producing active enzymes in cultured plant cells.

Effect of Inducers on the Production of 5‐Aminolevulinic Acid by Recombinant Escherichia coli

Aminolevulinic acid (ALA) was produced by recombinant Escherichia coli BL21(DE3) (pET28‐A.R‐hemA) harboring the ALA synthase gene (hemA) from Agrobacterium radiobacter zju‐0121. The effects of inducers on the ALA synthase activity and ALA productivity were evaluated. The results indicated that a low isopropyl‐β‐D‐thiogalactoside (IPTG) concentration (0.05 mmol/L) was favorable for high expression of ALA synthase, which resulted in higher ALA productivity. For metabolic engineering applications, lactose was a better substitute of IPTG for active enzyme expression. When lactose concentration was 5 mmol/L, the specific ALA synthase activity and ALA productivity reached 16.7 nmol/(min · mg of protein) and 1.15 g/L, respectively, which were about 15% and 43% higher than those induced by IPTG.

Expression of Active α-N-Acetylglucosaminidase/TAT Chimerae in Cultured Spodoptera frugiperda Cells

We examined the production and secretion of fusion constructs containing alpha-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/alpha-N-acetylglucosaminidase chimerae in enzyme replacement therapy.

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.

Relationship between Intron 4b Splicing of the Rat Geranylgeranyl Diphosphate Synthase Gene and the Active Enzyme Expression Level

Geranylgeranyl diphosphate synthase (GGPS) is a branch point enzyme in the mevalonate pathway that catalyzes the synthesis of geranylgeranyl diphosphate used for the geranylgeranylation of Rho, Rac and Rab proteins. The current study showed the production of multiple forms of GGPS mRNA from a single GGPS gene in rat. The mRNAs resulted from combinations of multiple alternative introns and two poly(A) sites in the 3'-translated and 3'-untranslated regions. These are classified into 1a-type and 1b-type mRNAs, based on the splicing of intron 4b resulting in the difference in deduced amino acid sequence between the C-terminal regions. The 1a-type and 1b-type proteins expressed in both Escherichia coli and HeLa cells were active and inactive, respectively. In the case of HeLa cells, the latter protein expression level was about 10% relative to the former one. This was also observed for Cos-7 and 293 cells. When fusions of beta-galactosidase with C-terminal regions differing between the 1a-type and 1b-type proteins were expressed in HeLa cells, the expressed fusion proteins were both found to be active but the latter fusion protein expression level was considerably low compared with the former one. The expression level of 1a-type mRNA was higher than that of 1b-type mRNA in brain, liver, heart, and thymus, but the two expression levels were the same in testis and ovary. During testis development the total GGPS mRNA expression level increased, accompanied by an increase in 1b-type mRNA, the expression level of 1a-type mRNA encoding active GGPS remaining kept unchanged. These results indicate that the expression level of rat active GGPS is at least regulated through the splicing of intron 4b of its gene.