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PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.
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6-Fitasa , Reactores Biológicos , Fermentación , Glucosa , Metanol , Proteínas Recombinantes , 6-Fitasa/genética , 6-Fitasa/metabolismo , Glucosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Metanol/metabolismo , Reactores Biológicos/microbiología , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/crecimiento & desarrollo , Pichia/genética , Pichia/metabolismo , Pichia/crecimiento & desarrollo , Expresión GénicaRESUMEN
OBJECTIVE: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics. METHODS: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay. RESULTS: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100ËC and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4ËC. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells. CONCLUSION: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.
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Conservación de Alimentos , Listeria monocytogenes , Pediocinas , Pediocinas/farmacología , Pediocinas/genética , Animales , Conservación de Alimentos/métodos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Humanos , Antibacterianos/farmacología , Clonación Molecular , Porcinos , Pruebas de Sensibilidad Microbiana , Bacteriocinas/farmacología , Bacteriocinas/genética , Bacteriocinas/metabolismo , Pediococcus acidilactici/genética , Pediococcus acidilactici/metabolismo , Expresión Génica , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/efectos de los fármacosRESUMEN
During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.
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COVID-19 , Saccharomycetales , Vacunas , Humanos , COVID-19/diagnóstico , COVID-19/prevención & control , Prueba de COVID-19 , Pichia/genética , Pichia/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Recombinantes/química , Vacunas/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos AntiviralesRESUMEN
Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.
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The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.
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Glicerol , Saccharomycetales , Glicerol/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Regiones Promotoras Genéticas , Carbono/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: ⢠Transient hypoxia and high methanol trigger high level of recombinant protein synthesis ⢠In Pichia pastoris, pAOX1 induction is higher in flocculent cells ⢠Medium heterogeneity leads to morphological diversification.
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Metanol , Pichia , Metanol/metabolismo , Pichia/genética , Pichia/metabolismo , Reactores Biológicos , Proteínas Recombinantes/metabolismo , HipoxiaRESUMEN
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
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Escorpiones , Ponzoñas , Animales , Humanos , Escorpiones/química , Ponzoñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismoRESUMEN
BACKGROUND: Although Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products. RESULTS: The expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h. At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level. CONCLUSION: We demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.
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Oligosacáridos , Azúcares , Oligosacáridos/metabolismo , Glucosa , Monosacáridos , Sacarosa/metabolismo , Fructosa/metabolismo , Fructanos/metabolismoRESUMEN
ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.
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Isópteros , beta-Glucosidasa , Animales , beta-Glucosidasa/química , Celobiosa/metabolismo , Isópteros/metabolismo , Pichia/metabolismo , Especificidad por Sustrato , Cinética , Glucosa/metabolismoRESUMEN
Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbß3, responsible for the platelet aggregation and αvß3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 µM ADP, 10 nM thrombin, and 1 µg/mL collagen (IC50 of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 µM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC50 1.77 µM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.
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Venenos de Crotálidos , Desintegrinas , Adhesión Celular , Movimiento Celular , Colágeno , Venenos de Crotálidos/química , Desintegrinas/química , Células Endoteliales , Humanos , Integrinas , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacologíaRESUMEN
Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.
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Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1-1/MPG1 genes rather than increased heterologous gene transcription.
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6-Fitasa , Técnicas de Cultivo Celular por Lotes , 6-Fitasa/genética , Expresión Génica , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , TemperaturaRESUMEN
Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.
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Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.
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Aspergillus oryzae , Aspergillus oryzae/genética , Hexosiltransferasas , Oligosacáridos , Pichia/genética , Saccharomycetales , SacarosaRESUMEN
Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.
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Probióticos , Saccharum , Thermoascus , Celulosa , Endo-1,4-beta Xilanasas/genética , Glucuronatos , Hidrólisis , Oligosacáridos , XilanosRESUMEN
Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain A. niger GH1 tannase, which was also produced in P. pastoris. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate's access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in P. pastoris, the tannase described could be used in food and beverage processing at low and medium temperatures.
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Aspergillus niger , Proteínas Fúngicas , Hidrolasas de Éster Carboxílico/química , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Pichia/genética , Pichia/metabolismo , SaccharomycetalesRESUMEN
The pandemics Corona Virus Disease started in 2019 (COVID-19) has being the third outbreak of coronaviruses in the last two decades. Initially associated to cases of pneumonia, the contamination was related to a seafood market in Wuhan City, China. The transmission of the virus occurs from person to person, directly or indirectly. SARS-Cov-2 belongs to the Coronaviridae family and the β-coronavirus genus. The virus genome is a single RNA strand positive of 30 kilobases (kb). The surface of the virus is composed of 3 main proteins, spike (S), membrane (M) and envelope (E). The receptor binding domain (RBD) is located in the S1 subunit. The viral RBD binds to angiotensina conversor enzyme (ACE 2) and then the virus can enter into the cells. The production of recombinant RBD becomes interesting as it can be used directly for the production of antibodies. The aim of this work is to produce the recombinant RBD of the S1 protein from SARS-Cov-2 for studies of immunological response. The yeast Pichia pastoris strains GS115 and Glycoswitch were used as expression systems, which implicates in different glycosylations of the antigen. GS115 determinates a glycosylation that can be beneficial as adjuvant, while the Glycoswitch strain, modified to reproduce humanized glycosylation, can induce a more specific immunity. The RBD protein was recovered from the supernatant and purified by metal affinity chromatography. The elution fractions were analyzed by SDS-PAGE and Western Blot. The yeast Pichia pastoris proved to be a good system to obtain the soluble recombinant RBD, but with low expression. New strategies to optimize the production are being evaluated. For example, a new strain of P. pastoris, X33 has been cloned to tests the expression and evaluate the yields.
A pandemia do Coronavírus iniciada em 2019 (COVID-19) foi o terceiro surto de coronavírus nas duas últimas décadas. Inicialmente associado a casos de pneumonia, a contaminação foi relacionada a um mercado de frutos do mar, na cidade de Wuhan, China. A transmissão do vírus ocorre de pessoa para pessoa, de forma direta ou indireta. O SARS-Cov-2 pertence à família Coronaviridae, do gênero β-corona vírus. O genoma do vírus é uma molécula única de RNA de fita positiva de 30 quilobases (kb). A superfície do vírus é composta por 3 proteínas principais, spike (S), membrana (M) e envelope (E). O domínio de ligação ao receptor (RBD) está localizado na subunidade S1 e liga-se ao receptor de enzima conversora de angiotensina (ACE 2), o que permite a entrada nas células. A produção de RBD recombinante torna-se interessante para utilização direta na produção de anticorpos. O objetivo deste trabalho é a produção recombinante do fragmento RBD da proteína S1 do vírus SARS-Cov-2 para estudos de resposta imunológica. A levedura Pichia pastoris cepas GS115 e Glycoswitch foram utilizadas como sistemas de expressão que preveem diferentes glicosilações do antígeno. A glicosilação determinada pela GS115 pode ter um resultado benéfico como adjuvante. A levedura Glycoswitch, modificada para reproduzir a glicosilação humanizada, pode ter um efeito benéfico produzindo resposta imune mais especifica. A proteína RBD foi recuperada no sobrenadante dos cultivos, purificada por cromatografia de afinidade a metal e as frações de eluição foram analisadas por SDS-PAGE e Western Blot. O sistema Pichia pastoris mostrou-se uma boa forma de expressão da proteína RBD recombinante solúvel, porém em baixa concentração. Novas formas de otimizar o rendimento estão sendo estudadas. Por exemplo, uma nova linhagem de P. pastoris, a X33, foi clonada, para testes de expressão e avaliação de rendimentos.
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Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO
Asunto(s)
Pichia/clasificación , Interferón beta/agonistas , Carcinoma Hepatocelular/patología , Optimización de Procesos , Codón , Células , Carcinoma Hepatocelular , Concentración de Iones de HidrógenoRESUMEN
Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.
Asunto(s)
Hexosiltransferasas/metabolismo , Oligosacáridos/metabolismo , Pichia/metabolismo , Alginatos/química , Carbohidratos/análisis , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Inmovilizadas , Fermentación , Hexosiltransferasas/genética , Humanos , Microbiología Industrial , Inulina/metabolismo , Melaza , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , Sacarosa , Tolueno/farmacología , Trisacáridos/biosíntesisRESUMEN
OBJECTIVE: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. RESULTS: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. CONCLUSION: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.