Bioprocess optimization of interferon ß-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

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

Optimization of SARS-CoV-2 spike protein receptor binding domain expression in Pichia pastoris and evaluation of its immunogenicity / 中华微生物学和免疫学杂志

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Immunogenicity of a Recombinant Gonadotropin-releasing Hormone Associated to the B Subunit of Escherichia coli Heat-labile Enterotoxin Expressed in Pichia pastoris and Escherichia coli Platforms

Abstract Gonadotropin-releasing hormone (GnRH) is one of the main targets for the development of immunocontraceptives vaccines. The aim of this study was to clone and express the recombinant GnRH fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) molecule in Pichia pastoris and Escherichia coli platforms and evaluate their immunogenicity in mice. P. pastoris (pGnRH/LTB) and E. coli (eGnRH/LTB) platforms were able to express GnRH/LTB expected band with ~ 21 kDa. Both constructions were immunogenic in mice. Similar IgG kinetics was observed for both construction when it was used as ELISA antigen respectively, showing significant (p<0.05) IgG levels 5-fold higher than a commercial vaccine and 14-fold higher than the controls. The histological effects of pGnRH/LTB as well as eGnRH/LTB proteins demonstrated a significant effect on the gonads, characterized by atrophy of seminiferous tubules, absence of spermatogenesis and reduction of Leydig cells. Both constructions were able to induce antibodies that block the hormone effect, suggesting the potential of GnRH/LTB, independently of the P. pastoris or E. coli platform used, as a vaccine candidate for immunocontraception.

Synthesis and antibacterial activity of C-7 haloacyl cephalosporins / 药学学报

Cephalosporins are widely used in the treatment of infectious diseases. The structural differences in cephalosporin drugs mainly lie in the C-7 amino side chain and the C-3 substituent. In this study, twenty-five haloacylated cephalosporins of five series were designed by using a strategy of introducing simple substituents at the C-7 amino group in four cephalosporin parent nucleus with different C-3 substituents and efficiently synthesized under optimized conditions. Their activities against human pathogenic bacteria, Pichia pastoris, citrus canker and citrus pathogenic fungi were evaluated. The results showed that most of the molecules had activity against human pathogenic bacteria, of which seven compounds including TM1f had stronger or equivalent inhibitory activities against eight human pathogens than the marketed drugs cefalotin, cefoxitin sodium and ceftizoxime sodium. The inhibitory activity of TM1s against Alternaria alternate Al.6 was stronger than that of cephalosporins and comparable to that of the positive control prochloraz. TM1f and TM1s are worthy of further study.

Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris / 生物工程学报

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Expression of / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by

Development and evaluation of a novel method for rapid screening of Pichia pastoris strains capable of efficiently expressing recombinant proteins / 生物工程学报

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

BACKGROUND: Methanol can be effectively removed from air by biofiltration (Shareefdeen et al., 1993; Babbitt et al., 2009 [1,2]). However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms (Negruta et al., 2010 [3]), and it can be released out of the cell constituting a secondary emission. RESULTS: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m−3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. CONCLUSIONS: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment.

Evaluation of Toxocara canis Glycosylated TES Produced in Pichia pastoris for Immunodiagnosis of Human Toxocariasis

Abstract Recombinant proteins are a suggested alternative for the diagnosis of toxocariasis. The current Escherichia coli recombinant protein overexpression system usually produces insoluble products. As an alternative, yeast such as Pichia pastoris have secretory mechanisms, which could diminish the cost and time for production. This study aimed to produce recombinant proteins in Pichia pastoris and verify their sensibility and specificity in an indirect ELISA assay. Two sequences (rTES-30 and rTES-120) of Toxocara canis excretory-secretory antigens were cloned in a pPICZαB vector and expressed in P. pastoris KM71H. Sera samples collected from human adults infected by Toxocara spp. were tested by indirect ELISA using rTES-30 and rTES-120 as antigens. Recombinant proteins were detected at 72 hours after induction, in the supernatant, as pure bands between 60~70 kDa with hyperglycosylation. Regarding diagnosis potential, recombinant antigens had high specificity (95.6%); however, sensitivity was 55.6% for rTES-30 and 68.9% for rTES-120. Further deglycosylation of the P. pastoris antigens did not seem to affect ELISA performance (p>0.05). The low sensitivity in the serodiagnosis diminished any advantage that P. pastoris expression could have. Therefore, we do not recommend P. pastoris recombinant TES production as an alternative for the diagnosis of toxocariasis.

Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate / 生物工程学报

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.

Expression of Ietalurus punetaus β-defensin based on recombinant Pichia pastoris / 生物工程学报

β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.

Impact of gene dosage on recombinant transglutaminase production of Pichia pastoris / 生物工程学报

Based on the rDNA sequence of Pichia pastoris, a multi-copy gene expression vector of transglutaminase (pPICZα-rDNA-mtg) was constructed and transformed to the host strain (pGAP9-pro/GS115) expressing pro peptide, to obtain the co-expression strain pro/rDNA-mtg (GS115). Real-time fluorescence quantitative PCR (qPCR) was used to analyze transglutaminase gene copy number in the 4 positive strains. We further studied the effect of gene copy on the enzyme production of recombinant Pichia pastoris as well as high-density fermentation of higher expression strain in a 3-L fermenter. The mtg copy numbers of the 4 positive strains were 2.21, 3.36, 5.72 and 7.62 (mtg-2c, mtg-3c, mtg-6c and mtg-8c), respectively, and the enzyme production capacity and protein expression level were mtg-3c>mtg-2c>mtg-6c>mtg-8c. Mtg-3c and mtg-6c of high-density fermentation had the highest enzymatic activity and enzymatic activity per unit wet weight in the supernatant of 3.12 U/mL, 52.1 U/g (wet weight) and 2.07 U/mL and 36.5 U/g (wet weight), respectively. In terms of enzyme activity per unit wet weight, mtg-3c is 1.4 times higher than that of mtg-6c. The activity of purified enzyme (mtg-3c) was up to 7.21 U/mL and the protein concentration was 437.2 μg/mL. By analyzing the effect of mtg copy number on the enzyme production of recombinant strains, mtg-3c is suitable for the co-expression of two genes (pro and mtg) in pro/rDNA-mtg, and its enzyme activity is related to higher protein secretion of the strain.

Optimization of enterokinase secretion in Pichia pastoris / 生物工程学报

Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences. Therefore, enterokinase has been widely used as a tool enzyme in the field of biomedicine. Currently, the expression level of enterokinase in Pichia pastoris is low, which hinders related practical applications. In this study, the effects of six different signal peptides SP1, SP2, SP3, SP4, SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied. Compared with α-factor, SP1 significantly increased the secretory expression of enterokinase (from 6.8 mg/L to 14.3 mg/L), and the enterokinase activity increased from (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±489) U/mL by co-expressing the endogenous protein Kex2. Moreover, the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Background: Methanol can be effectively removed from air by biofiltration. However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms, and it can be released out of the cell constituting a secondary emission. Results: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m− 3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. Conclusions: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment

High-cell density cultivation of recombinant Pichia pastoris harboring fungal endoinulinase and prebiotics applications of inulo-oligosaccharides

Aim: The objective of the present study was to clone and express A. niger endoinulinase gene in P. pastoris for high-level expression. Further to explore high cell density cultivation, biochemical characterization of recombinant endoinulinase and application of inulo-oligosaccharides (IOS) as prebiotics was also studied. Methodology: Molecular cloning of A. niger endoinulinase gene in P. pastoris, screening of positive clones by genomic DNA PCR, shake flask studies, high cell density fermentation performed with both conventional and temperature shift approach, biochemical characterization of endoinulinase and in-vitro fermentation of IOS was carried out to confirm prebiotic efficacy. Results: The endoinulinase gene of 1482 bp from Aspergillus niger was genetically engineered in the GS115 host and was secreted extracellularly using α signal sequence. As a result of fermentation with the conventional approach, recombinant endoinulinase activity was enhanced to 65.7 U ml-1. Recombinant endoinulinase showed absolute substrate specificity for inulin, hydrolyzing inulin to IOS with the DP range 3-4. Interpretation: Hydrolysis of inulin by recombinant endoinulinase was characterized. In-vitro fermentation of IOS by lactic acid and bifidogenic bacteria was studied as a part of industrial application and functional properties of IOS was similar to commercial prebiotics.

Expressão da L-asparaginase II de Saccharomyces cerevisiae recombinante extracelular com glicosilação humanizada em Pichia pastoris / Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris with humanized glycosylation

L-asparaginase é um inibidor eficiente do crescimento tumoral, usado em sessões de quimioterapia contra a Leucemia Linfoblástica Aguda (LLA), resultando na remissão completa da doença em 90% dos pacientes tratados. A L-asparaginase II de Saccharomyces cerevisiae (ScASNaseII) tem alto potencial de superar os efeitos adversos da L-asparaginase de bactéria, porém sua produção endógena resulta em uma proteína hipermanosilada e, consequentemente, imunogênica. A cepa de Pichia pastoris Glycoswitch tem a maquinaria para expressar e secretar altas quantidades de enzima com glicosilação humanizada. Nesse trabalho, descrevemos o processo genético para expressar a ScASNaseII no meio extracelular pela P. pastoris Glycoswitch, e também os parâmetros bioquímicos, perfil cinético, citotoxicidade contra células leucêmicas e a interferência da glicosilação na atividade da enzima obtida. Nossos dados mostram que a cepa aplicada foi capaz de expressar ScASNaseII no meio extracelular passível de purificação de proteínas contaminantes com apenas um passo cromatográfico. A atividade específica para asparagina foi 218,2 UI/mg e a atividade glutaminásica representou 3,1% da atividade asparaginásica. Os parâmetros cinéticos foram KM = 120,5 µM e a eficiência catalítica de 3,8 x 105 M-1s-1. Análises por meio de gel nativo sugerem uma conformação tetramérica de aproximadamente 150 kDa. Essa é uma nova estratégia de produzir essa enzima de forma extracelular, com mais facilidade de purificação e com melhores propriedades biotecnológicas

L-asparaginase is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cell; its use results in 90% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to overcome the side effects of bacteria L-asparaginase, but the endogenous production of it results in hypermannosylated immunogenic enzyme. However, Pichia pastoris Glycoswitch strain has the machinery to express and secrete high quantity of the enzyme and with humanized glycosylation. Here we describe the genetic process to acquire the ScASNaseII in the extracellular medium expressed by P. pastoris Glycoswitch, and the biochemical properties of the resultant enzyme, kinetic profile, cytotoxicity against ALL cell line and the interference of glycosylation in its activity. Our data show that the strain employed is able to express extracellular asparaginase active and possible to be purified of contaminant proteins using a single chromatographic step. The specific activity using asparagine was 218.2 IU.mg-1 and the glutaminase activity represents 3.1% of its asparaginase activity. The kinetics parameters were KM=120.5 µM and a catalytic efficiency of 3.8x105 M-1s-1. The Native-PAGE suggested a tetrameric protein conformation, with approximately 150 kDa. This is a novel strategy to produce this enzyme extracellularly, easier to purify and with better biotechnological properties

Expressão e caracterização da glicoproteína D do HSV-1 geneticamente fusionada às oncoproteínas E6 e E7 do HPV-16 e HPV-18 (gDE7E6) em células de mamífero / Expression and characterization of the genetically fused HSV-1 glycoprotein D to E6 and E7 oncoproteins HPV-16 e HPV-18 (gDE7E6) in mammalian cells

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais

Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes

High expression and immunogenicity analysis of HA globular head domain of H5 subtype avian influenza virus produced in Pichia pastoris / 生物工程学报

To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218) and the optimal dosage of the recombinant H5HA was 50 μg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.

Expression of Pichia pastoris tRNAProCCG and its function / 生物工程学报

Translocation ribonucleic acid (tRNA) is one of the important components in protein synthesis. In order to explore the effect of the changes of tRNAs corresponding to rare codons (rarity tRNAs) on the expression of exogenous genes, the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed. The expression of GFP in P. pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene. The expression amount of the repressed GFP could be increased about 4.9% when tRNAProCCG gene was cointegrated to the 3' of the repressed GFP gene through pPIC9K to the genome of P. pastoris GS115. Meanwhile, the expression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNAProCCG gene to the genome of P. pastoris GS115 through pPIC9K and pFLDα, respectively. Using the same method, NFATc3T-GFP fusion gene and tRNAProCCG gene were co-expressed in P. pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein. In conclusion, tRNAProCCG gene has been confirmed to be a kind of rare tRNAs in P. pastoris GS115. Through co-expression of tRNAProCCG gene and heterologous genes which containing the continuous rare codon CCG, the expression of the repressed heterologous genes could be increased significantly. Furthermore, this co-expression system would contribute to screening and determining the other rare tRNAs.

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity / 生物工程学报

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.