Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

Secretory Expression of Mycoplasma hyopneu-moniae P97R1 Gene in Pichia pastoris and Primary Application of the Expression Product

[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.

Expression and Purification of Arabidopsis High Mobility Group B Protein Gene At2G34450 in Pichia pastoris

[Objective] The aim was to study the expression of Arabidopsis gene A/2G34450 in Pichia pastoris and to obtain recombinant Arabidopsis HMGB protein. [Method] The At2G34450 gene was cloned into yeast expression vector pPIC9K containing AOXl promoter and the sequences of secreting α-signal peptides. Recombinant plasmid was linearized by Sal l and transformed into P. pastoris GSl15 competent cells by electroporation. Positive integrated clones were screened out, and the At2G34450 protein was expressed under the induction of methanol. [Result] The At2G34450 protein was expressed in yeast medium through methanol induction. SDS-PAGE results showed that recombination product was At2G34450 protein. [Conclusion] At2G34450 protein was successfully expressed in the P. pastoris system for the first time, which paves a direct path to further research on the functions of HMGB family members.

Induction and expression of T4 lysozyme gene in Pichia pastoris

Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The recombinant plasmid was digested by Sal I and then introduced into prepared GS115 competent cells by electroporation. Positive clone and multiple inserts were screened. The secreted proteins in the supernatants were tested. In the agar holes diffusion assay, our expressed protein showed significant antibacterial circles. Results T4 lysozyme protein inhibited the growth of staphylococcus aureus and streptococcus Pneumoniae. There was no difference in the bactericidal activity and the amount of protein expression between the single and multiple copies. The antibacterial activity of expressed protein remained the same during the heat stability test. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. There is no relationship between copy number and expression. T4 lysozyme protein is heat stable.

Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris

The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use tot industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects： （1） increase recombinant cell concentrations in fermentor to high density during growth phase; （2） effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; （3） decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.

Expression of Recombinant Human Lysozyme-tachyplesin I(hLYZ-TP I)in Pichia Pastoris and Analysis of Antibacterial Activity

Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Expression and Characterization of a Thermostable Xylanase Gene xynA from a Themophilic Fungus in Pichia pastoris

The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, which coded 194 amino acids. The putative amino acid sequence and DNA sequence of xylanase from T. lanuginosus SY2 （GenBank no.： GU166389） were 98.97 and 99.49% identical to the other T. lanuginosus （GenBank no.： U35436）. A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. The recombinant strain was identified by G418 selection and confirmed by PCR analysis. It was induced by 1.0% methanol at 28°C to express the recombinant xylanase. The results showed that the recombinant xylanase was secreted into extracellular fermentation liquid. The highest enzyme activity of 113.5 IU mL-1 and protein content of 889.7 μg mL-1 were detected for 216 h of induction. The optimal pH value and temperature of the enzyme activity was 5.5 and 65°C, respectively. The xylanase activity retained above 80% from pH value 2.5 to 8.5 for 48 h. The enzyme activity was above 85% at incubation temperature of 55°C.

Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.

Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris

To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes（850 amino acids） from Guangxi in China was inserted into Pichia pastoris expression vector pPICZαB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pastoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA.

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.

Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Constitutive and Secretory Expression of the AiiA in Pichia pastoris Inhibits Amorphophallus konjac Soft Rot Disease

Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) which is generated by Bacillus species has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain recombinant yeast which produces AiiA protein. The recombinant yeast Pichia pastoris GS115 was constructed to constitutive expression of the AiiA gene. The results of reverse transcript PCR analysis showed that the AiiA gene was expressed successfully in the yeast. Proteins extracted from YPDS showed the highest inhibition efficacy to E. carotovora compared with the other two mediums (YPD and LB) under tested conditions.

An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.

Codon Optimization of SMAP-29 Gene and Its Expression in Pichia pastoris

[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.

Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

Construction and expression of a synthetic gene encoding nonstructural glycoprotein NS1 of dengue 2 virus in Pichia pastoris

To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.

Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris

The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.

Expression and Characterization of HGV E2 cDNA in Pichia pastoris

A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was inserted intoPichia pastoris expression vector pPIC9K in the reading frame of α-factor secreting signal peptide. The recombinant expression plasmid pPIC9K-E2 was introduced intoP. pastoris GS 115 with electroporation and recombined with the host genome by homological recombination. The His+Mut+ recombinant yeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0.5% methanol, the target protein (E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 protein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with sera from the immunized rabbits and the patients infected by GBV-C/HGV.