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)

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.

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.

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.

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.

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.

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.

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

Oligomerization triggered by foldon to enhance the catalytic efficiency of feruloyl esterase / 生物工程学报

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.

Development of a purification tag to produce thermostable fused protein / 生物工程学报

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.

Characterization and structure of a novel thermostable glucoamylase from Talaromyces leycettanus JCM12802 / 生物工程学报

Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3±24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA's thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.

Heterologous expression and characterization of Aspergillus oryzae acidic protease in Pichia pastoris / 生物工程学报

Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn²⁺ and Cu²⁺ enhanced the activity of PepA, whereas Fe³⁺, Fe²⁺ and Ca² had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.

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.

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.

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.