Pichia Expression Research Articles

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin.

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (K d for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris: A case study with proteolysis prone streptokinase

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.

Systemic and sustained thioredoxin analogue prevents acute kidney injury and its-associated distant organ damage in renal ischemia reperfusion injury mice

The mortality of patients with acute kidney injury (AKI) remains high due to AKI associated-lung injury. An effective strategy for preventing both AKI and AKI-associated lung injury is urgently needed. Thioredoxin-1 (Trx) is a redox-active protein that possesses anti-oxidative, anti-apoptotic and anti-inflammatory properties including modulation of macrophage migration inhibitory factor (MIF), but its short half-life limits its clinical application. Therefore, we examined the preventive effect of a long-acting Trx, which is a fusion protein of albumin and Trx (HSA-Trx), against AKI and AKI-associated lung injury. Recombinant HSA-Trx was expressed using a Pichia expression system. AKI-induced lung injury mice were generated by bilateral renal ischemia reperfusion injury (IRI). HSA-Trx administration attenuated renal IRI and its-associated lung injury. Both renal and pulmonary oxidative stress were suppressed by HSA-Trx. Moreover, HSA-Trx inhibited elevations of plasma IL-6 and TNF-α level, and suppressed IL-6–CXCL1/2-mediated neutrophil infiltration into lung and TNF-α-mediated pulmonary apoptosis. Additionally, HSA-Trx suppressed renal IRI-induced MIF expression in kidney and lung. Administration of HSA-Trx resulted in a significant increase in the survival rate of renal IRI mice. Collectively, HSA-Trx could have therapeutic utility in preventing both AKI and AKI-associated lung injury as a consequence of its systemic and sustained multiple biological action.

The endemic GII.4 norovirus-like-particle induced-antibody lacks of cross-reactivity against the epidemic GII.17 strain.

Norovirus‐like particle (VLP) vaccine is promising against human norovirus infection. Unfortunately, genetic diversity of norovirus hindered the development of this vaccine. In this study, the immunogenicity of norovirus VLPs induced by the endemic GII.4 and the epidemic GII.17 genotypes, and the cross‐reactivity between them as well as GI.1 and GII.3 VLPs were evaluated in mice by using serum IgG and histo‐blood group antigen (HBGA) blocking antibodies as index. Results showed well immunogenicity of both GII.4 and GII.17 VLPs in mice. Serum IgG GMT (Geometric Mean Titer) were 3.63 (GII.4) and 3.88 (GII.17) respectively, and sustained to the 15th week. The HBGA blocking antibodies were 130 (GII.4) and 360 (GII.17) respectively at the end of the 4th week. Additionally, there was a dramatically statistical difference found in the cross‐reactivity within genogroup (GII.3, GII.4 and GII.17) (p < .001), and also showed similar difference between genogroups (GI.1 vs. GII.3, GII.4 and GII.17) (p < .001). Summarized the pPICZa pichi pichia expression system showed a potential to be the alternative for expression of norovirus VLPs in secretion form, and the little cross‐reactivity found between the endemic strain and the epidemic strain provides an evident for the consideration of selecting candidates of norovirus vaccine strains.

Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels. We identified two different CCE04 alleles in both resistant strains, CCE04SR-VP and CCE04London , with CCE04SR-VP being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity. Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04SR-VP being a likely hypothesis. © 2019 Society of Chemical Industry.

Characterization of a highly stable \u03b1-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system

BackgroundStructurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study.ResultsReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively.ConclusionA modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.

A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

In this study, we identified a P450 enzyme (STH10) and an oxidoreductase (POR) from Thanatephorus cucumeris NBRC 6298 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol was performed by using Pichia pastoris whole cells coexpressing sth10 and por, and the product analysis indicated that the STH10 enzyme possessed steroidal 19- and 11β-hydroxylase activities. This is a novel fungal P450 enzyme with 19-hydroxylase activity, which is different from the known steroidal aromatase cytochrome P450 19 (CYP19) and CYP11B families of enzymes.IMPORTANCE Hydroxylation is one of the most important reactions in steroid functionalization; in particular, C-19 hydroxylation produces a key intermediate for the synthesis of 19-nor-steroid drugs without a C-19 angular methyl group in three chemoenzymatic steps, in contrast to the current industrial process, which uses 10 chemical reactions. However, hydroxylation of the C-19 angular methyl group remains a very challenging task due to the high level of steric resistance to the C-19 methyl group between the A and B rings. The present report describes a novel fungal P450 enzyme with 19-hydroxylase activity. This opens a new venue for searching effective biocatalysts for the useful process of steroidal C-19 hydroxylation, although further studies for better understanding of the structural basis of the regioselectivity and substrate specificity of this fungal steroidal 19-hydroxylase are warranted to facilitate the engineering of this enzyme for industrial applications.

Secretion expression of human neutrophil peptide 1 (HNP1) in Pichia pastoris and its functional analysis against antibiotic-resistant Helicobacter pylori.

Human neutrophil peptide 1 (HNP1) is a small (3.44kDa) cationic peptide that is a distinct member of the defensin family. HNP1 plays a crucial role in controlling bacterial infections, particularly by antibiotic-resistant bacteria, through membrane perforation patterns. The structural characteristics of HNP1's three intramolecular disulfide bridges cause difficulty in its synthesis via chemical methods. In this study, bioactive recombinant HNP1 was produced using the Pichia pastoris (P. Pichia) expression system. HNP1 was fused with the polyhedrin of Bombyx mori and enhanced green fluorescent protein (EGFP) to prevent HNP1 toxicity in yeast host cells under direct expression. An enterokinase protease cleavage site (amino acid sequence DDDDK) was designed upstream of the HNP1 peptide to obtain the antibacterial peptide HNP1 with native structure after it was cleaved by the enterokinase. The fusion HNP1 protein (FHNP1) was successfully expressed and had a molecular mass of approximately 62.6kDa, as determined using SDS-PAGE and Western blot. Then, the recovered FHNP1 was digested and purified; Tricine-SDS-PAGE results showed that HNP1 was successfully released from FHNP1. Functional analysis of induction against antibiotic-resistant Helicobacter pylori (H. pylori) showed that it was challenging for HNP1 to acquire resistance to the antibiotic-resistant H. pylori. Moreover, in vitro studies showed that HNP1 exerted a strong effect against antibiotic-resistant H. pylori activity. Furthermore, the animal model of H. pylori infection established in vivo showed that HNP1 significantly reduced the colonization of antibiotic-resistant H. pylori in the stomach. Our study indicated that this could be a new potential avenue for large-scale production of HNP1 for therapeutic application against the antibiotic-resistant H. pylori infection in humans.

Strategies for cloning and expression of a codon‐optimized human P‐glycoprotein (MDR1) in the yeast Pichia pastoris

P‐glycoprotein (P‐gp) is an ABC transporter which actively transports xenobiotics out of cells. Overexpression of P‐gp is one of the main causes for the development of multidrug resistances (MDR) in cancer and other diseases. Because of its ability to bind and transport a large variety of chemically different compounds, overexpression of P‐gp in these MDR cancers leads to resistances against chemotherapeutic drugs that are distinctly different in structure, chemical composition, and functionality. Using high‐throughput in silico ligand docking studies, we have previously identified drug‐like compounds that inhibit P‐gp and cause reversal of MDR in a prostate cancer cell line (Brewer et al., Mol. Pharmacol. 86, 716–726, 2014; Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). To analyze the mechanism of inhibition by these initially identified P‐gp inhibitors as well as newly discovered drug‐like compounds that reversed MDR in cancers, biochemical and biophysical analysis of inhibitor action is imperative. For this reason, a gene for human P‐gp (MDR1) was designed with optimized codon usage for translation in P. pastoris. A pPinkHC‐MDR1 plasmid was created for integration into Pichia pastoris PichiaPink™, a commercially available high yield expression system that includes specific strains with protease knock outs to limit protein degradation. Successful MDR1 DNA integration in the PichiaPink™ chromosome was confirmed, but significant protein expression has thus far not been observed. We have re‐cloned the codon optimized MDR1 DNA into another Pichia expression vector, pHIL. This vector allows integration of the gene of interest into the Pichia pastoris strain, GS115, which has been used successfully by us in the past to express a mouse cysteine‐less P‐glycoprotein. The resulting colonies displayed a Muts phenotype, which is indicative of the correct recombination into the Pichia genomic DNA. Initial Western blot analyses using both P‐gp specific and His‐tag specific antibodies showed that after 24 hours of growth in minimal methanol media the MDR1 GS115 clone did express reasonable amounts of human P‐gp. We report here our attempts at constructing a stable, codon optimized, human P‐gp gene for protein production in Pichia pastoris as well as attempts to express, purify and characterize the human P‐glycoprotein.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, Ms. Lynn McBee and New England Biolabs, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High Catalytic Current Density Based on Direct Bioelectrocatalysis of a PQQ Domain of Pyranose Dehydrogenase

Introduction A pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH) is an eukaryotic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase consisting three-domains structure [1]. The PQQ and heme b cofactors are located in the 45 kDa and 21 kDa domains, respectively, and a small C-terminal domain is family 1-type carbohydrate-binding module, which these domains are connected by a proline-rich linker region. CcPDH shows the oxidation activity toward monosaccharides in a 1C4 chair conformation such as D-glucosone and L-fucose. The N-terminal cytochrome domain is a 6-coordinated low-spin heme b with Met/His ligands that enables direct electrical contact between the enzyme and the electrode [2]. Electrons are transferred from reduced PQQ in catalytic domain to heme b in cytochrome domain. The previous study reported that intact CcPDH is capable of direct electron transfer (DET)-based bioelectrocatalysis on a glassy carbon (GC) electrode [3]. The catalytic currents were observed at a lower potential than the redox potential of heme b in the cytochrome domain, suggesting that both the PQQ and the cytochrome domains in CcPDH are able to DET with the GC electrode. In present study, to examine DET reaction of the PQQ domain, an isolated PQQ domain was expressed in Pichia expression system. Here, we demonstrated the direct bioelectrocatalysis for the isolated PQQ domain from C. cinerea pyranose dehydrogenase. The bioelectrocatalytic current density of 1.6 mAcm-2 was achieved on a preformed 2-mercaptoethanol (ME) self-assembled monolayer (SAM) modified gold nanoparticles (AuNPs) electrode under optimized conditions. Experimental The direct bioelectrocatalysis of DHPDH was obtained by using glassy carbon (GC) electrode modified with the enzyme as working electrode. An aliquot of 30 μL of 1 μM enzyme solution containing 10 μM PQQ and 1 mM CaCl2 was dropped on the entire surface of the GC electrode and then dried at room temperature. AuNPs-modified and unmodified polycrystalline Au electrodes were used for the direct bioelectrocatalysis of DHPDH. 1 μL of the concentrated AuNPs was dropped onto the surface of the Au electrode, and then air-dried. To prepare electrodes with immobilized DHPDH, AuNPs-modified and unmodified polycrystalline Au electrodes were immersed in 20 mM aqueous solution of 2-mercaptoethanol for 1 h at room temperature (ME-AuNPs electrode and ME-Au electrode). Then they were immersed in 1 μM holo-DHPDH in a 50 mM sodium acetate buffer solution (pH 6.0) at 4 ℃ for over 15 h. Cyclic voltammetry measurements were performed in 50 mM various pH buffer with 100 mM L-fucose. A conventional three-electrode cell was used, in which a platinum wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode. Results and Discussion Initially, a clear catalytic current of L-fucose oxidation by the DHPDH on the GC electrode was observed starting at same potential of intact CcPDH. It is demonstrated that the DHPDH can also directly transfer electrons to the electrodes. The catalytic current density of DHPDH (0.9 μA cm-2, at 0.35 V) was an order of magnitude smaller than that of intact CcPDH. To improve the direct bioelectrocatalysis of DHPDH, a thiol-based SAM coated Au electrodes were studied. Accordingly, the optimal SAM for DHPDH was found to be shorter alkyl chain length and hydroxide-functionalized thiols. The catalytic current density of 11 μA cm-2 (at 0.35 V) was obtained when using ME-Au electrode. DHPDH immobilized on ME-Au electrode showed optimum at pH 6.0 and a value of K m = 35.1 mM, respectively. In order to obtain higher catalytic currents, furthermore, AuNPs were cast on the surface of Au electrode. Increasing the number of AuNP casts from 3 to 15 times on led to increasing catalytic current densities from 125 μA cm-2 to 240 μA cm-2 at 0.35 V. Additionally, we had considered the effects of the electrolyte (ion conductivity) and temperature dependence for catalytic current by AuNPs-Au electrode at the casting cycle three times. The catalytic current at 50 °C was approximately 5-times higher than that at 20 °C. Upon increasing acetate buffer concentration from 50 mM to 500 mM, the current density was enhanced to as 1.7-fold. Figure 1 shows the cyclic voltammogram of L-fucose oxidation on DHPDH-modified ME-AuNPs electrode under optimized conditions of 50 °C, pH 6, 500 mM acetate buffer, and the AuNPs casting cycle 15 times. Finally, the catalytic current reached to a steady-state value of 1.6 mA cm-2.

Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins

The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins.

Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis.

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development.

Efficient Isolation, Expression and Improvement Technique System for Feed Enzymes in China

Supplementation of hydrolytic enzymes in feed can decrease the effect of anti‐nutritional factors and increase feed conversion rate by 5‐10%. Consequently, this practice can improve the efficiency of livestock production and reduce environmental contamination. The authors have developed a highly efficient platform to enrich the genetic resources of several enzymes, characterize and assess their application potentials, and achieve high‐yield production. These works make the production of novel enzymes cost‐effective and commercially valuable.Firstly, a new technology system was set up, which can directly clone full‐length genes and screen objective genes from environmental metagenomic or transcriptomic nuclear acid. Using these methods, 184 genes coding for enzymes have been identified in short time. The enzymes showed different properties including high catalytic efficiency, thermophilic, acidophilic, or protease‐resistance. Secondly, great contributions were made in the structure and function relationship research of enzyme proteins. An efficient screening vector system was constructed based on extracellular fusion mutant proteins, optimized the protein engineering system for enzyme improvement, and obtained several improved enzymes with excellent properties and application potentials. Third, another great finding was identification of several key factors in Pichia expression system and verification of their function. Based on these knowledge, a high‐yield expression system of 5‐50 mg/mL was developed for large‐scale, cost‐effective production of enzymes, including phytase, xylanase, glucanase, mannannase, and α‐galactosidase.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris

Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.

High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation

article i nfo Background: Lysozyme plays a crucial role in innate immunity with its well-recognized bacteriolytic activity. In this study, the influence of expression parameters (inoculation volume, culture volume, growth time, induction temperature and time, initial pH and methanol concentration) on human lysozyme (HLZ) production in recombinant P. pastoris SMD1168 was investigated through Plackett-Burman (PB) design and response surface methodology (RSM). Results: It was revealed that induction temperature, induction time and culture volume had significant influence (P b 0.01) on HLZ expression level, which were elected for further optimization with three-dimensional response surface designs for enhanced HLZ production. The highest lysozyme activity reached 3301 U/mL under optimized conditions (at 23.5°C for 90 h with culture volume of 48 mL) in shake flask, which increased 2.2 fold compared with that achieved with the standard protocol (Invitrogen). When high-cell-density fermentation of the recombinant Pichia pastoris was performed in a 15 L fermenter under optimized conditions, the extracellular lysozyme activity reached 47,680 U/mL. SDS-PAGE analysis of the product demonstrated that HLZ was produced as a single major protein with a molecular weight of approximately 14.7 kDa, consistent with its expected size. Conclusions: The results indicated that the optimized culture conditions using PB design and RSM significantly enhanced the expression level of HLZ, and the Pichia expression system for HLZ production was successful and industrially promising.

Genetic transformation to integrate two expression cassettes into the genome of yeast Pichia pastoris

Background Pichia pastoris is a methylotrophic species of yeast, which means that it can grow on methanol as its sole carbon and energy source [1]. The Pichia expression system has several advantages: short length of the oligosaccharide chains added to proteins, correct folding and very high cell densities [2]. P. pastoris vectors are designed for homologous integration into either AOX I locus, one of the two homologous AOX I genes present in this species or his4 locus. Gene insertion events at the AOX I (GS115) loci arise from a single crossover events between the loci and any of the three AOX I regions in the vector, the AOX I promoter, the AOX I transcription termination region (TT), or sequences even further downstream of AOX I (3′ AOX I) [3]. In either GS115 gene insertion events at his4 locus arise from a single crossover event between his4 locus in the chromosome and His4 gene of the vector. This results in the insertion of one or more copies of the vector in his4 locus [3].

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

A simple strategy for the generation of multi-copyPichia pastoriswith the efficient expression of mannanase

Pichia pastoris expression system was widely used for producing heterologous proteins. Some researches revealed that the increase in the copy number could improve the expression of foreign genes in P. pastoris. Hundreds or thousands of antibiotic-resistance recombinants need to be screened because the frequency of multiple gene insertion events is very low in P. pastoris. The traditional method of constructing multi-copy gene is complicated, tedious, and full of randomness. Here, we developed a rapid method for constructing multi-copy Pichia expression vectors harboring mannanase gene. The developed strategy is easy to manipulate genetically and can precisely generate plasmids with a certain copy number of heterologous gene. The average mannanase activities of recombinants randomly chosen from two-, four-, and six-copy recombinant libraries were 1.7-, 2.2-, and 1.3-folds, respectively, of that from single-copy recombinant library. The result revealed that the strategy could effectively improve the expression of foreign proteins in P. pastoris.

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3' ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.