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

Usos de la levadura Pichia pastoris en la producción de proteínas recombinantes / Uses of Pichia pastoris yeast in the production of recombinant proteins

La levadura metilotrófica Pichia pastoris (clasificada actualmente como Komagataella phaffii) es una de las más importantes para la producción de proteínas heterólogas. En el trabajo se presenta un análisis de las principales características que se ponen de manifiesto en la expresión de proteínas recombinantes expresadas en este microorganismo. Se describen las cepas disponibles para la transformación y producción de proteínas recombinantes expresadas en Pichia pastoris, los principales vectores comerciales para la expresión, los promotores más eficientes, los marcadores seleccionables, la señal de secreción, los métodos usados en las transformaciones genéticas y los patrones de glicosilación que se presentan. Se brindan recomendaciones generales acerca de los parámetros de bioprocesos como la composición del medio, el pH, la temperatura, la velocidad de aireación, la inducción y las estrategias de alimentación para alcanzar altos valores de productividad. Se presentan los resultados de las aplicaciones de Pichia pastoris en la producción de dos vacunas en Cuba, la vacuna contra la hepatitis B y la vacuna para el control de la garrapata(AU)

Pichia pastoris metylotrofic yeast (currently classified as Komagataella phaffii) is one of the most important yeast for the production of heterologous proteins. The work presents an analysis of the main characteristics that are marked in the production of recombinant proteins expressed in Pichia pastoris. It describes the strains available for the transformation and production of recombinant proteins expressed in P. pastoris, the main commercial vectors for expression, the most efficient promoters, selectable markers, the secretion signal, the methods used in genetic transformations and glycosylation patterns that occur. General recommendations are provided on bioprocess parameters such as media composition, pH, temperature, aeration velocity, induction, and feeding strategies to achieve high productivity values. The results of Pichia pastoris applications for the production of two vaccines in Cuba, the hepatitis B vaccine and the tick control vaccine are shown(AU)

Mejora tecnológica del proceso de llenado aséptico para la producción de la vacuna Antihepatitis B recombinante / Improvement of the aseptic filling process for the production of recombinant hepatitis vaccine

Frente a la creciente demanda de producción de la vacuna Antihepatitis B recombinante, constituye un reto para el Centro Nacional de Biopreparados aumentar la fabricación del producto, para lo cual el proceso de llenado aséptico requirió de una inversión. En el trabajo se presenta la selección de la nueva máquina llenadora y se calculan los indicadores económicos asociados a la inversión, la que se recupera en el cuarto año con una ganancia de $2.655.300. Luego de la inversión se evaluó el desempeño de la nueva máquina y se comparó con los resultados anteriores a la inversión. Se compararon los valores de volumen dispensado por vial, velocidad de llenado, rendimiento operacional, principales defectos detectados en los lotes de llenado, tiempo promedio de llenado de un lote, costo de producción y comportamiento del monto resarcido al cliente por rechazos de producto. El volumen dispensado por vial resulta más exacto, reduciendo las pérdidas de producto. La velocidad de llenado aumenta 1,7 veces respecto a la máquina anterior. El rendimiento operacional aumenta en un 13,63 percent. Disminuyen los rechazos de producto en 40.897 viales, representando un ahorro de $24.538 ingresados en 73 lotes producidos. Se ahorra en energía eléctrica un total de $14.718 en un mes. El costo unitario del proceso de llenado disminuye en 0,0648 $/vial(AU)

National Center for Biopreparations must increase the production of the recombinant hepatitis B vaccine because of its growing demand. In order to fulfill this challenge, the aseptic filling process required an investment. This work, presents the selection of the new filling machine and the economic indicators that support the investment. Inversion cost is recovered in the fourth year with a profit equivalent to $2,655,300. After the investment, the performance of the new machine was compared with the previous one. Volume dispensed per vial, filling speed, operational performance, major defects detected in filling batches, average filling time for a batch, production cost and payments to customers due to rejected products were compared. The control of the volume dispensed per vial is more accurate, reducing product losses. The filling speed increases 1.7 times compared to the previous machine. Operational performance increases by 13.63 percent. Product rejections are reduced by 40,897 vials, saving $24,538 for the 73 batches. Electricity consumption diminished, saving $14,718 monthly. The unit cost of the filling process decreases by $0.0648/vial(AU)

Advances in metabolic engineering of non-conventional yeasts / 生物工程学报

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

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.

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.

Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

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.

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

Levedura antagonista no controle de fungos filamentosos deteriorantes de alimentos / Antagonistic yeast against deteriorating mold of food

Este trabalho avaliou o efeito antifúngico in vitro de toxina killer de Hansenula wingei contra Aspergillus ochraceus e Penicillium expansum deteriorantes de alimentos. O extrato livre de células-ELC contendo a toxina killer (obtido a partir do cultivo da levedura em Caldo MPL a 25ºC/96 horas) foi submetido ao ensaio antifúngico por meio de análise microscópica, determinando-se a porcentagem de germinação conidial e o desenvolvimento de hifas dos fungos testados. H. wingei inibiu a germinação conidial de A. ochraceus e P. expansum em 98,91% e 96,49%, respectivamente, bem como a inibição do desenvolvimento micelial de ambos os fungos foi maior que 78%. O composto antifúngico mostrou-se estável ao tratamento térmico de 90ºC/30 min., indicando a possibilidade de aplicação no biocontrole in situ de frutos pós-colheita.

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

Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris / 生物工程学报

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.

Fermentation optimization based on cell self-adaptation to environmental stress - a review / 生物工程学报

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.

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.

Preparation and characterization of recombinant human-source collagen / 生物工程学报

This study aimed to obtain a recombinant human-source collagen for industrialization. First, based on the Gly-X-Y sequence of human type I collagen, we optimized the hydrophilic Gly-X-Y collagen peptide, designed the human collagen amino acid sequence and the corresponding nucleotide sequence. Next, the expression vector pPIC9K-COL was constructed via endonuclease digestion technology. We obtained an engineering strain of human-source collagen by electrotransforming Pichia pastoris, and then it was fermented, purified and identified. As a result, the expression level reached 4.5 g/L and the purity was over 95%. After amino acid N-terminal sequencing, molecular weight analysis, amino acid analysis and collagenase degradation test, we confirmed that the obtained collagen was consistent with designed primary structure of human-source collagen. After freeze-drying, we analyzed the collagen by scanning electron microscope and cell cytotoxicity, confirming that the collagen has porous fiber reticular structure and superior cytocompatibility. This indicates that human-source collagen has potential to be applied as biomedical material. In conclusion, we successfully obtained the expected human-source collagen and laid a foundation to its further application.