High-yield expression of recombinant mouse coagulation factor Ⅶ in methylotrophic yeast Pichia pastoris

Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.

Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α 1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.

High-level secretory expression, purification, and characterization of an anti-human Her II monoclonal antibody, trastuzumab, in the methylotrophic yeast <i>Pichia pastoris</i>

DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man9-GlcNAc2, Man10-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.

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 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.

Optimization of Media for Production of an Effective Yeast Biocontrol Agent Pichia membranefaciens

The growth of Pichia membranefaciens was studied using different nitrogen and carbonsources as substrates. Among nitrogen sources tested, soya peptone, yeast extract power,beef extract and polypeptone were relatively favorable to the growth of yeast. Thedensity of the yeast showed to be directly proportional to carbon sources supplementation.Glucose and fructose were good carbon sources for the yeast growth. However, lactoseshowed poor performance for the cell growth of the yeast. In this study, beef extractpresented a good synergic effect on the yeast growth with different carbonhydrates. Themedium for P.membranefaciens used glucose and beef extract as substrates. The higherconcentration of glucose and beef extract, the better growth of P.membranefaciens. Theaddition of chlorella growth factor (CGF) stimulated markedly the growth of P.membranefaci-ens.The increased concentration of CGF from 0.5 to 1% did not enhance the numbers ofP.membranefaciens. This result will help design a better strategy for scale-up produc-tion of P.membranefaciens.

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.

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.

人成骨蛋白成熟肽基因在毕氏酵母(Pichia Yeast)中的高效表达

采用PCR方法,根据文献报道的人成骨蛋白（osteogenic protein-1,OP-1）成熟肽基因序列,设计并合成一对引物,从构建的含人成骨蛋白基因的质粒中扩增获得大小为420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,并以pPIC9K为表达载体构建重组表达质粒,转化大肠杆菌细胞,经鉴定的阳性重组质粒并线形化,电转化毕氏酵母细胞GS115,于30℃进行甲醇诱导分泌表达,表达产物存在于培养基中,占分泌蛋白的10％.重组表达产物进行Western Blot可以检测到重组表达产物,ELISA检测其具有特异性结合活性.

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein(HPV16 L1) in Pichia pastoris

In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.

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.

Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

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 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.

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.