RESUMEN
Voltage-gated sodium channels are blocked by local anesthetic and anticonvulsant drugs. A receptor site for local anesthetics has been defined in transmembrane segment S6 in domain IV (IVS6) of the alpha subunit, but the anticonvulsant lamotrigine and related compounds have more complex structures than local anesthetics and may interact with additional amino acid residues. Apparent K(D) values for inactivated-state block of rat brain type IIA sodium channels expressed in Xenopus oocytes were 31.9 micro M, 17.3 micro M, 3.7 micro M and 10.3 micro M for lamotrigine and compounds 227c89, 4030w92 and 619c89, respectively. Compound 619c89 was the strongest frequency-dependent blocker, which correlated with higher affinity and a five-fold slower recovery from drug block compared to lamotrigine. Examination of lamotrigine block of mutant sodium channel alpha subunits, in which alanine had been substituted for each individual amino acid in IVS6, identified mutations I1760A, F1764A and Y1771A as causing the largest reductions in affinity (six-, seven- and 12-fold, respectively). The ratios of effects of these three mutations differed for compounds 227c89, 4030w92, and 619c89. The amino acid residues interacting with these pore-blocking drugs define a surface of IVS6 that is exposed to the pore and may rotate during gating.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Estructura Terciaria de Proteína/efectos de los fármacos , Agonistas de los Canales de Sodio , Triazinas/farmacología , Alanina/genética , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Activación del Canal Iónico/efectos de los fármacos , Cinética , Lamotrigina , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Inhibidores de la Captación de Neurotransmisores/farmacología , Oocitos , Técnicas de Placa-Clamp , Piperazinas/farmacología , Estructura Terciaria de Proteína/fisiología , Pirimidinas/farmacología , Proteínas Recombinantes/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Triazinas/química , XenopusRESUMEN
Voltage-gated sodium channels consist of a pore-forming alpha subunit and two auxiliary beta subunits. Excitable cells express multiple alpha subtypes, designated Na(v)1.1-Na(v)1.9, and three beta subunits, designated beta1, beta2 and beta3. Understanding how the different alpha subtypes, in combination with the various beta subunits, determine sodium channel behavior is important for elucidating the molecular basis of sodium channel functional diversity. In this study, we used whole-cell electrophysiological recording to examine the properties of the human Na(v)1.3 alpha subtype, stably expressed in Chinese hamster ovary cells, and to investigate modulation of Na(v)1.3 function by beta1, beta2 and beta3 subunits. In the absence of beta subunits, human Na(v)1.3 formed channels that inactivated rapidly (tau(inactivation) approximately equals 0.5 ms at 0 mV) and almost completely by the end of 190-ms-long depolarizations. Using an intracellular solution with aspartate as the main anion, the midpoint for channel activation was approximately -12 mV. The midpoint for inactivation, determined using 100-ms conditioning pulses, was approximately -47 mV. The time constant for repriming of inactivated channels at -80 mV was approximately 6 ms. Coexpression of beta1 or beta3 did not affect inactivation time course or the voltage dependence of activation, but shifted the inactivation curve approximately 10 mV negative, and slowed the repriming rate ca. three-fold. beta2 did not affect channel properties, either by itself or in combination with beta1 or beta3. Na(v)1.3 expression is increased in damaged nociceptive peripheral afferents. This change in channel expression levels is correlated with the emergence of a rapidly inactivating and rapidly repriming sodium current, which has been proposed to contribute to the pathophysiology of neuropathic pain. The results of this study support the hypothesis that Na(v)1.3 may mediate this fast sodium current.
Asunto(s)
Canales de Sodio/biosíntesis , Animales , Células CHO/metabolismo , Cricetinae , Canales Epiteliales de Sodio , Humanos , Potenciales de la Membrana/fisiología , Canales de Sodio/química , Canales de Sodio/fisiologíaRESUMEN
Studies with animal seizure models have indicated that changes in temporal and spatial expression of voltage-gated sodium channels may be important in the pathology of epilepsy. Here, by using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes [Whitaker et al. (2000) J. Comp. Neurol. 422, 123-139], we have compared the cellular expression of all four brain alpha-subunit sodium channel mRNAs in "normal" and epileptic hippocampi from humans. Neuronal cell loss was observed in all regions of the hippocampus of diseased patients, indicating that sclerosis had occurred. Losses of up to 40% compared to post-mortem controls were observed which were statistically significant in all regions studied (dentate gyrus, hilus, and CA1-3). To assess mRNA levels of the different alpha-subtypes in specific subregions, control and diseased tissue sections were hybridised to subtype-specific probes. To quantify any changes in expression while allowing for cell loss, the sections were processed for liquid emulsion autoradiography and grain counts were performed on populations of individual neurones in different subregions. No significant differences were found in the expression of type I and VI mRNAs. In contrast, a significant down-regulation of type II mRNA was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71+/-7% of control, P<0.01), CA2 (81+/-8% of control, P<0.05) and CA1 (72+/-6% of control, P<0.05) compared with control tissue. Additionally, a significant up-regulation in type III mRNA in epileptic CA4 pyramidal cells (145+/-7% of control, P<0.05) was observed. It is not clear whether these changes play a causal role in human epilepsy or whether they are secondary to seizures or drug treatment; further studies are necessary to investigate these alternatives. However, it is likely that such changes would affect the intrinsic excitability of hippocampal neurones.
Asunto(s)
Epilepsia/metabolismo , Hipocampo/metabolismo , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Canales de Sodio/genética , Adolescente , Anciano , Epilepsia/genética , Epilepsia/fisiopatología , Femenino , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Hibridación in Situ/métodos , Masculino , Potenciales de la Membrana/genética , Persona de Mediana Edad , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Sondas de Oligonucleótidos , Células Piramidales/patologíaRESUMEN
Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel alpha subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the alpha subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human alpha subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.
Asunto(s)
Química Encefálica , Canales de Sodio/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Especificidad de Anticuerpos , Femenino , Humanos , Activación del Canal Iónico , Masculino , Potenciales de la Membrana , Persona de Mediana Edad , Neuritas/química , Conejos , Canales de Sodio/inmunologíaRESUMEN
The human brain voltage-gated Na+ channel type IIA alpha subunit was cloned and stably expressed in Chinese hamster ovary cells and its biophysical and pharmacological properties were studied using whole-cell voltage-clamp. Fast, transient inward currents of up to -8,000 pA were elicited by membrane depolarization of the recombinant cells. Channels activated at -50 mV and reached maximal activation at -10 mV to 0 mV. The reversal potential was 62 +/- 2 mV which is close to the Na+ equilibrium potential. The half-maximal activation and inactivation voltages were -24 +/- 2 mV and -63 +/- 1 mV, respectively. Currents were reversibly blocked by tetrodotoxin with a half-maximal inhibition of 13 nM. The effects of four commonly used anti-convulsant drugs were examined for the first time on the cloned human type IIA channel. Lamotrigine and phenytoin produced concentration- and voltage-dependent inhibition of the type IIA currents, whereas, sodium valproate and gabapentin (up to 1 mM) had no effect. These results indicate that recombinant human type IIA Na+ channels conduct tetrodotoxin-sensitive Na+ currents with similar properties to those observed in recombinant rat brain type IIA and native rat brain Na+ channels. This stable cell line should provide a useful tool for more detailed characterization of therapeutic modulators of human Na+ channels.
Asunto(s)
Química Encefálica , Expresión Génica , Canales de Sodio/genética , Canales de Sodio/fisiología , Animales , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , Conductividad Eléctrica , Electrofisiología , Humanos , Lamotrigina , Técnicas de Placa-Clamp , Fenitoína/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Tetrodotoxina/farmacología , Transfección , Triazinas/farmacologíaRESUMEN
Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na(+) channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an alpha-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na(+) channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na(+) channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.
Asunto(s)
Anestésicos Locales/farmacología , Anticonvulsivantes/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales de Sodio/química , Canales de Sodio/metabolismo , Sustitución de Aminoácidos/genética , Anestésicos Locales/metabolismo , Animales , Anticonvulsivantes/metabolismo , Sitios de Unión , Encéfalo , Electrofisiología , Etidocaína/metabolismo , Etidocaína/farmacología , Lamotrigina , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Piperazinas/metabolismo , Piperazinas/farmacología , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Pirimidinas/metabolismo , Pirimidinas/farmacología , Ratas , Bloqueadores de los Canales de Sodio , Canales de Sodio/genética , Termodinámica , Triazinas/metabolismo , Triazinas/farmacología , Xenopus laevisRESUMEN
This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins. The protocols listed here for S. cerevisiae are for three widely used types of promoter: galactose-regulated (GAL1, GAL7, GAL10), glucose-repressible (e.g., ADH2), and constitutive glycolytic (e.g., PGK or GAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol-regulated AOX1 promoter and HIS4 selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small-scale S. cerevisiae and P. pastoris cultures and also for high-density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided for S. cerevisiae and yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high-density cultures (e.g., 130 g/liter). Finally, a Support Protocol describes small-scale preparation of protein extracts.
Asunto(s)
Proteínas Recombinantes de Fusión/metabolismo , Levaduras/genética , Fermentación , Expresión Génica , Vectores Genéticos/genética , Pichia/genética , Pichia/crecimiento & desarrollo , Pichia/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Transformación Genética , Levaduras/crecimiento & desarrollo , Levaduras/metabolismoRESUMEN
The type III voltage-gated sodium channel was cloned from human brain. The full-length cDNA has 89% identity with rat type III, and the predicted protein (1951 amino acids) has 55 differences. The expression pattern of human type III mRNA was determined in adult brain tissue and, in contrast to rat, was detected in many regions, including caudate nucleus, cerebellum, hippocampus and frontal lobe. The human type III channel was stably expressed in Chinese hamster ovary (CHO) cells and its biophysical properties compared to the human type II channel using identical conditions. The voltage dependence and kinetics of activation were found to be similar to that of type II. The kinetics of inactivation of the two human subtypes were also similar. However, type III channels inactivated at more hyperpolarized potentials and were slower to recover from inactivation than type II. When expressed in human embryonic kidney (HEK293T) cells, type III channels produced currents with a prominent persistent component, which were similar to those reported for rat type II [Ma et al. (1997) Neuron, 19, 443-452]. However, unlike type II, this was prominent even in the absence of coexpressed G-proteins, suggesting type III may adopt this gating mode more readily. The distinct properties of the channel, together with its wide distribution in adult brain, suggest that in humans, type III may have important physiological roles under normal, and perhaps also pathological conditions.
Asunto(s)
Encéfalo/metabolismo , Canales de Sodio/química , Canales de Sodio/fisiología , Médula Espinal/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , Proteínas de Unión al GTP/metabolismo , Humanos , Riñón , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Sodio/genética , Tetrodotoxina/farmacología , Transcripción Genética , TransfecciónRESUMEN
Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. Application of 10 microM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between -80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of -63 mV under control conditions to -85 mV in the presence of 100 microM retigabine (IC(50) = 5.2 microM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.
Asunto(s)
Anticonvulsivantes/farmacología , Carbamatos/farmacología , Fenilendiaminas/farmacología , Canales de Potasio/metabolismo , Animales , Células CHO , Cricetinae , Electrofisiología , Humanos , Canal de Potasio KCNQ2 , Canal de Potasio KCNQ3 , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje , Transfección , XenopusRESUMEN
The distribution of mRNAs encoding voltage-gated sodium channel alpha subunits (I, II, III, and VI) and beta subunits (beta1 and beta2) was studied in selected regions of the human brain by Northern blot and in situ hybridisation experiments. Northern blot analysis showed that all regions studied exhibited heterogenous expression of sodium channel transcripts. In situ hybridisation experiments confirmed these findings and revealed a predominantly neuronal distribution. In the parahippocampal gyrus, subtypes II and VI and the beta-subunit mRNAs exhibited robust expression in the granule cells of the dentate gyrus and pyramidal cell layer of the hippocampus. Subtypes I and III showed moderate expression in granule cells and low expression in the pyramidal cell layer. Distinct expression patterns were also observed in the cortical layers of the middle frontal gyrus and in the entorhinal cortex. In particular, all subtypes exhibited higher levels of expression in cortical layers III, V, and VI compared with layers I and II. All subtypes were expressed in the granular layer of the cerebellum, whereas specific expression of subtypes I, VI, beta1, and beta2 mRNAs was observed in Purkinje cells. Subtypes I, VI, and beta1 mRNAs were expressed, at varying levels, in the pyramidal cells of the deep cerebellar nuclei. These data indicate that, as in rat, human brain sodium channel mRNAs have a distinct regional distribution, with individual cell types expressing different compliments of sodium channels. The differential distribution of sodium channel subtypes suggest that they have distinct roles that are likely to be of paramount importance in maintaining the functional heterogeneity of central nervous system neurons.
Asunto(s)
Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , ARN Mensajero/metabolismo , Canales de Sodio/metabolismo , Animales , Humanos , Activación del Canal Iónico , RatasRESUMEN
The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/fisiología , Saccharomyces cerevisiae/fisiología , Humanos , Feromonas/fisiologíaRESUMEN
After transection of their axons within the sciatic nerve, DRG neurons become hyperexcitable. Recent studies have demonstrated the emergence of a rapidly repriming tetrodotoxin (TTX)-sensitive sodium current that may account for this hyperexcitability in axotomized small (<27 microm diam) DRG neurons, but its molecular basis has remained unexplained. It has been shown previously that sciatic nerve transection leads to an upregulation of sodium channel III transcripts, which normally are present at very low levels in DRG neurons, in adult rats. We show here that TTX-sensitive currents in small DRG neurons, after transection of their peripheral axonal projections, reprime more rapidly than those in control neurons throughout a voltage range of -140 to -60 mV, a finding that suggests that these currents are produced by a different sodium channel. After transection of the central axonal projections (dorsal rhizotomy) of these small DRG neurons, in contrast, the repriming kinetics of TTX-sensitive sodium currents remain similar to those of control (uninjured) neurons. We also demonstrate, with two distinct antibodies directed against different regions of the type III sodium channel, that small DRG neurons display increased brain type III immunostaining when studied 7-12 days after transection of their peripheral, but not central, projections. Type III sodium channel immunoreactivity is present within somata and neurites of peripherally axotomized, but not centrally axotomized, neurons studied after <24 h in vitro. Peripherally axotomized DRG neurons in situ also exhibit enhanced type III staining compared with control neurons, including an accumulation of type III sodium channels in the distal portion of the ligated and transected sciatic nerve, but these changes are not seen in centrally axotomized neurons. These observations are consistent with a contribution of type III sodium channels to the rapidly repriming sodium currents observed in peripherally axotomized DRG neurons and suggest that type III channels may at least partially account for the hyperexcitibility of these neurons after injury.
Asunto(s)
Ganglios Espinales/fisiología , Neuronas/fisiología , Nervio Ciático/fisiología , Canales de Sodio/genética , Raíces Nerviosas Espinales/fisiología , Secuencia de Aminoácidos , Animales , Axones/fisiología , Femenino , Laminectomía , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Rizotomía , Nervio Ciático/lesiones , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Raíces Nerviosas Espinales/lesiones , Tetrodotoxina/farmacología , Regulación hacia ArribaRESUMEN
Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.
Asunto(s)
Clonación Molecular/métodos , Expresión Génica , Productos del Gen env/biosíntesis , Vectores Genéticos , Gentamicinas/farmacología , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Secuencia de Bases , Northern Blotting , Western Blotting , Farmacorresistencia Microbiana , Productos del Gen env/aislamiento & purificación , Genes Fúngicos , Proteína gp120 de Envoltorio del VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pichia/efectos de los fármacos , Pichia/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Transformación GenéticaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/genética , VIH-1 , Pichia/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Vectores Genéticos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción GenéticaAsunto(s)
ADN Recombinante/genética , Regulación Fúngica de la Expresión Génica , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Levaduras/genética , Secuencia de Aminoácidos , Vectores Genéticos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded greater than 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Bordetella pertussis/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Proteínas Recombinantes/biosíntesis , Factores de Virulencia de Bordetella , Tos Ferina/prevención & control , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Secuencia de Bases , Western Blotting , Fermentación , Expresión Génica , Vectores Genéticos/genética , Datos de Secuencia Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797-802]. In addition, the full-length protein, mEGF1-53, was secreted by P. pastoris.
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Pichia/genética , Proteínas de Saccharomyces cerevisiae , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Clonación Molecular , Factor de Crecimiento Epidérmico/aislamiento & purificación , Factor de Crecimiento Epidérmico/metabolismo , Proteínas Fúngicas/genética , Cinética , Ratones , Pichia/metabolismo , Plásmidos , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.