Yeast Pichia Pastoris Research Articles

Overcoming multidrug resistance in cancer cells: Biochemical evaluation of new lead drug candidates

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer cells due to its role in the transport of chemotherapeutic drugs out of the cells in an ATP‐dependent process. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds. To assess functionality of the hit compounds we measured their effects on P‐gp ATPase hydrolysis activity. Some of the compounds inhibited ATP hydrolysis of purified P‐gp and successfully reversed the MDR in prostate and ovarian cancer cell lines. We subsequently used computational approaches and structure‐based design to produce variants of one of the hit compounds with predicted higher affinity to P‐gp. The efficacy of these variants in reversing MDR in cell culture as well as the biochemical evaluation of inhibition was assessed. High expression of human P‐gp in the yeast Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Currently, reconstitution of P‐gp into nanodiscs allowed for human P‐gp to be solubilized in its active form with ATPase activity enhanced by 2–3 fold as well as increased stability. Here we report the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

Optimizing Protein Function for Screening of Novel Inhibitors that Target Multi‐Drug Resistance in Cancers

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of proteins and is strongly linked with multidrug resistance (MDR) in tumors where overexpression often abolishes the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. To evaluate the mechanism of inhibition in human P‐gp, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. In order to effectively assess the potential inhibitors, we must be able to sufficiently stimulate our protein and have a high enough activity to see the inhibitory effects of the potential inhibitors. In previous work, we found that the activity of P‐gp assembled into membrane nanodiscs had significantly higher basal activity in the absence of transport substrate than P‐gp in detergent micelles. However, significant increase in activity in the presence of the substrate verapamil was not observed in micelles or nanodiscs. Here, we report methods of protein purification and nanodisc assembly modified to have optimally functional human P‐gp. The resulting purified P‐gp will be used to evaluate the inhibition mechanism of lead compounds that were shown to reverse MDR in cancer cell culture.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples, Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

Expression of an α-KTx from Tityus serrulatus scorpion venom in Pichia pastoris yeast and its preliminary electrophysiological characterization

Expression of an α-KTx from Tityus serrulatus scorpion venom in Pichia pastoris yeast and its preliminary electrophysiological characterization

STUDY OF RECOMBINANT PROTEIN FRACTIONS FROM THE BOMBYX MORI AND PICHIA PASTORIS USING "NATIVE PAGE" METHOD

We have studied the recombinant protein fractions of ̴ 24-34 kDa obtained from Bombyx mori - silkworm larvae and yeast Pichia pastoris. Fractions, containing the recombinant proteins by gel-electrophoresis using the “Native PAGE” method, which allows for differentiation of proteins according to their natural (surface) charge and conformational properties were studied. The results showed that the recombinant protein fraction from Bombyx mori (̴ 34 kDa) consisted of four components and fraction from Pichia pastoris (̴ 24 kDa) consisted one component. The most optimal conditions for the “Native PAGE”- the main gel: Tris HCl (pH-8.8) 244 mM, Bis-Acrylamide 10%, TEMED 0.08%, APS 0.08%; concentration gel: Tris HCl (pH-6.8) 125 mM, Bis-Acrylamide 4%, TEMED 0.05%, APS 0.05% were found.

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications.

The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut− phenotype cannot grow on methanol as a single carbon source. We assessed the Mut− phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (PAOX1) was active in the Mut− phenotype and showed adequate eGFP fluorescence levels and protein yields (YP/X) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using PAOX1HSA and PAOX1vHH expression constructs. Scenario B comprising a glucose‐methanol phase and a 72‐hr‐long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut− phenotype, the oxygen demand, heat output, and specific methanol uptake (q methanol) in the methanol phase were reduced by more than 80% compared with the MutS phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut− strain for current and future applications in the field of recombinant protein production.

ER arrival sites associate with ER exit sites to create bidirectional transport portals.

COPI vesicles mediate Golgi-to-ER recycling, but COPI vesicle arrival sites at the ER have been poorly defined. We explored this issue using the yeast Pichia pastoris. ER arrival sites (ERAS) can be visualized by labeling COPI vesicle tethers such as Tip20. Our results place ERAS at the periphery of COPII-labeled ER export sites (ERES). The dynamics of ERES and ERAS are indistinguishable, indicating that these structures are tightly coupled. Displacement or degradation of Tip20 does not alter ERES organization, whereas displacement or degradation of either COPII or COPI components disrupts ERAS organization. We infer that Golgi compartments form at ERES and then produce COPI vesicles to generate ERAS. As a result, ERES and ERAS are functionally linked to create bidirectional transport portals at the ER-Golgi interface. COPI vesicles likely become tethered while they bud, thereby promoting efficient retrograde transport. In mammalian cells, the Tip20 homologue RINT1 associates with ERES, indicating possible conservation of the link between ERES and ERAS.

The cloning of the virus envelope glycoprotein F of canine distemper virus expressed in Pichia pastoris

Canine distemper virus (CDV) is a pathogen which affects members of the Canidae family, causing an acute, often fatal, systemic disease. CDV is an RNA virus of the family Paramyxoviridae that contains two envelope glycoproteins: F and HA. In this study, we focused on the envelope glycoprotein F as the main target for neutralizing antibodies produced after infection or vaccination. The complete coding region of the protein (60 kDa) was expressed in the methylotrophic yeast Pichia pastoris, obtained in a recombinant form and secreted to the culture medium. Later, to analyze its immunogenicity, the protein was combined with an oily adjuvant and used to inoculate mice. The results provide evidence supporting a potential application of this recombinant protein as a subunit vaccine.

N-Phenylbenzamide derivatives as alternative oxidase inhibitors: Synthesis, molecular properties, 1H-STD NMR, and QSAR

In the present work, 117 N-phenylbenzamides (NPDs) were prepared and evaluated against recombinant AOX from the fungal pathogen Moniliophthora perniciosa. 1H, 13C NMR, FTIR, and mass spectra provided structural information on NPDs. The library compounds were tested as Alternative Oxidase inhibitors in two different assays using the model yeast Pichia pastoris: cell growth and oxygen consumption assays. The most active compound, 3FH, was further characterized by DRX and 1H-NMR-STD. Single crystal X-ray diffraction showed intra- and intermolecular interactions of 3FH in solid-state and elucidated its 3D structural configuration. 1H-NMR-STD allowed us to derive protein-ligand interactions in a membrane-mimetic system and evidenced an outstanding interaction of 3FH with this enzyme. Results of both biological assays were used as input to Quantitative Structure-Activity Relationship models, which highlighted the more important molecular fragments contributions for protein-ligand interaction.

Engineering and optimization of phosphate-responsive phytase expression in Pichia pastoris yeast for phytate hydrolysis

Phytate is the major storage form of phosphorus in plants. It is present in cereals and raw materials of vegetable origin used in animal and human diets. However, non-ruminant animals have little phytase activity in their guts and, therefore, cannot digest phytate. As a result, almost all dietary phytate is discharged into the environment, causing phosphorus pollution. Phytate is also considered as an “antinutrient” for its ability to form insoluble and stable complexes with metal ions, thus reducing dietary absorption of essential minerals. It is a dire need to develop sustainable approaches for environmentally-friendly utilization for this valuable and abundant natural resource. To this end, we engineered Pichia pastoris to express and secrete phytase in a “made-to-order” fashion in response to external level of inorganic phosphate (Pi). Responsiveness to external Pi level was achieved by generating a Pi-responsive promoter library using directed evolution. The resultant yeast strains were proven to liberate Pi from wheat-based meal in a simulated in vitro digestion model. These yeast-based whole cell biocatalysts may serve as platform hosts with potential applications in food processing industry and animal waste treatment.

Engineering a Pichia pastoris nitrilase whole cell catalyst through the increased nitrilase gene copy number and co-expressing of ER oxidoreductin 1.

1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

Expression of Functional Human Na+- Coupled Citrate Transporter (SLC13A5) in the Yeast Pichia pastoris

Expression of Functional Human Na+- Coupled Citrate Transporter (SLC13A5) in the Yeast Pichia pastoris

Purification and Stability Expression of Open Reading Frames encoding Fusion and Non Fusion Human Interferon Alpha-2a produced in Methilotropic Yeast Pichia pastoris

Recombinant human interferon alpha-2a (hIFNα-2a) is therapeutic protein that widely used in hepatitis B/C and several cancer treatments. We developed higher molecular weight of hIFNα-2a to improve protein pharmacokinetic profile. The protein was designed as a fusion protein with human serum albumin as protein tag. The protein was produced in Pichia pastoris with 85 kDa in size. This research was aimed to purify, characterize and determine the stability expression of open reading frame (ORF) encoding Fusion and Non fusion forms of hIFNα-2a. The proteins were purified using affinity chromatography and characterized using SDS PAGE and Western Blotting methods. Protein recovery yield was determined by ELISA. Stability expression was applied in generation time until 90th generation. The results showed that the Fusion and Non fusion proteins were successfully purified with 74-79% of protein recovery. The proteins can be recognized by specific monoclonal antibody and verified as hIFNα-2a Fusion and Non fusion with 85 kDa and 19 kDa in size respectively. The expression stability showed that the proteins were still produced in Pichia pastoris until 90th generation time with no significant difference of expression level. To conclude, the expression level of ORFs encoding Fusion and Non fusion hIFNα-2a was stable until 90th generations.

Recombinant expression of an oriental river prawn anti-lipopolysaccharide factor gene in Pichia pastoris and its characteristic analysis

Anti-lipopolysaccharide factors (ALFs), as main host-defense molecules of crustaceans, form a unique family of antimicrobial peptides (AMPs). MnALF4 is one isoform of ALFs isolated from the freshwater prawn Macrobrachium nipponense. In the present study, MnALF4 gene was successfully expressed in the yeast Pichia pastoris and the recombinant MnALF4 protein exhibited efficient and broad-spectrum antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria in vitro. When prawns were injected with rMnALF4 before bacterial challenge with E. coli, the recombinant protein effectively promote the elimination of bacteria by the host. It manifested that rMnALF4 could effectively kill the invading bacteria in vivo. Treatment with rMnALF4 led to remarkable changes in bacterial morphology, such as spheroidization, oversized bacteria, and cell lysis. In addition, rMnALF4 showed weak hemolysis activity to the rabbit red blood cells. Our work suggests that MnALF4 plays an important role in Macrobrachium immunity and is worthy of further investigation as a potential antibacterial agent with high efficacy against bacterial infection and low toxicity to host cells.

Effect of ergosterol on the interlamellar spacing of deuterated yeast phospholipid multilayers

Sterols regulate several physico-chemical properties of biological membranes that are considered to be linked to function. Ergosterol is the main sterol molecule found in the cell membranes of yeasts and other fungi. Like the cholesterol found in mammalian cells, ergosterol has been proposed to have an ordering and condensing effect on saturated phospholipid membranes. The effects of cholesterol have been investigated extensively and result in an increase in the membrane thickness and the lipid acyl chain order. Less information is available on the effects of ergosterol on phospholipid membranes.Neutron Diffraction (ND) was used to characterize the effect of ergosterol on lipid multilayers prepared with deuterated natural phospholipids extracted from the yeast Pichia pastoris. The data show that the effect of ergosterol on membranes prepared from the natural phospholipid extract rich in unsaturated acyl chains, differs from what has been observed previously in membranes rich in saturated phospholipids. In contrast to cholesterol in synthetic phospholipid membranes, the presence of ergosterol up to 30 mol % in yeast phospholipid membranes only slightly altered the multilayer structure. In particular, only a small decrease in the multilayer d-spacing was observed as function of increasing ergosterol concentrations. This result highlights the need for further investigation to elucidate the effects of ergosterol in biological lipid mixtures.

Synthesis and characterisation of magnetic κ-carrageenan nanocomposites for chitinase33 enzyme immobilisation

Chitinases are chitin-degrading enzymes that have wide biotechnological applications in the fields of medicine, agriculture and the industry. Production of chitinase on an industrial scale requires high protein expression. Pichia pastoris yeast is an important host for the rapid production and high levels of recombinant proteins. In this project, the fungal chit33 enzyme was expressed and purified in Pichia pastoris X-33, and the purified enzyme was immobilised to enhance the sustainability, activity, and reuse on the magnetite κ-carrageenan/chitosan nanocomposite as a biocatalyst. The synthesised nanoparticle was characterised using FTIR, SEM and EDS analysis. According to the results of enzyme activity measurement under different pH conditions, the temperature and time of the stabilised enzyme showed better activity and stability than the free enzyme. Compared to free enzyme and chitosan beads, in the magnetic κ-carrageenan/chit36 substrate the specific activity, pH tolerance, optimum temperature of the enzyme was improved. The enzyme stabilised in the magnetic cariogenic/chit33 substrate at 70°C maintains about 80% of its enzyme activity relative to the magnetic chitosan/chit36 bead and maintains about 40% of its activity after eight cycles of re-use of the enzyme.

Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc Transporter.

The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Synthesis and characterisation of magnetic κ-carrageenan nanocomposites for chitinase33 enzyme immobilisation

Chitinases are chitin-degrading enzymes that have wide biotechnological applications in the fields of medicine, agriculture and the industry. Production of chitinase on an industrial scale requires high protein expression. Pichia pastoris yeast is an important host for the rapid production and high levels of recombinant proteins. In this project, the fungal chit33 enzyme was expressed and purified in Pichia pastoris X-33, and the purified enzyme was immobilised to enhance the sustainability, activity, and reuse on the magnetite κ-carrageenan/chitosan nanocomposite as a biocatalyst. The synthesised nanoparticle was characterised using FTIR, SEM and EDS analysis. According to the results of enzyme activity measurement under different pH conditions, the temperature and time of the stabilised enzyme showed better activity and stability than the free enzyme. Compared to free enzyme and chitosan beads, in the magnetic κ-carrageenan/chit36 substrate the specific activity, pH tolerance, optimum temperature of the enzyme was improved. The enzyme stabilised in the magnetic cariogenic/chit33 substrate at 70°C maintains about 80% of its enzyme activity relative to the magnetic chitosan/chit36 bead and maintains about 40% of its activity after eight cycles of re-use of the enzyme.

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

ABSTRACT The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts.

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.