High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

Expression, Identification and Characterize of CD25-Binding Epitope Modified Human IL-2 in Pichia pastoris

Interleukin-2 (IL-2) plays important roles in variety of immune functions and it is widely used in the medication. But in recent years it was reported that vascular leak syndrome (VLS) was induced by IL-2. Evidences showed that the interaction of IL-2 and IL-2Rαβγ (CD25) caused VLS. Thus, this experiment modified the CD25-binding epitope in human IL-2 (hIL-2) to minimize the side effect of IL-2 in the medication. In this study, a recombinant human interleukin 2 (rhIL-2) was expressed in Pichia (P.) pastoris. An effective strategy was established to express rhIL-2 protein in 120 L scale and the optimal purification procedure was investigated. The purity of rhIL-2 in final product was about 98 % and the concentration of the rhIL-2 was 0.45 mg/mL. Bioactivity analysis showed that the purified rhIL-2 protein displayed high activity on proliferation of CTLL-2 cells and increased the ratio of CD4⁺/CD8⁺. It indicates that the target protein is expressed and the character of the rhIL-2 has high activity. This study provides a strategy for large-scale production of bioactive rhIL-2 protein using P. pastoris as an expression host.     

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.     

Expression,purification and characterization of recombinant human interleukin-22 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Interleukin-22 (IL-22) is a member of the IL-10 family. Its potential in clinical use has been highlighted for its important roles in promoting antimicrobial defense and preventing epithelial damages. Previous studies have reported that IL-22 can be expressed using prokaryotic systems and purified from inclusion bodies, however the recovery rate was poor. To produce functional IL-22 with a high yield, human IL-22 was inserted into the eukaryotic expression vector pPICZαA and transformed into Pichia pastoris. The expression of recombinant human IL-22 (rhIL-22) was induced by methanol and accounted for about 85% of the total secreted proteins. A simple purification strategy was established to purify the rhIL-22 from the culture supernatant, yielding 100 mg/l at 90% purity by chromatography with a SP Sepharose FF column. Bioactivity analysis showed the purified rhIL-22 demonstrated a specific activity that was comparable with the commercial one. This study provides a new strategy for large-scale production of bioactive IL-22 for use in basic studies and therapeutic applications.     

Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris

A DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280 mg L(-1) rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170 mg L(-1) . Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20,908.85 Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies.     

High yield and purification of recombinant human apolipoprotein E3 in Pichia pastoris

Apolipoprotein E3 (ApoE3) is an important apolipoprotein in plasma and plays a critical role in lipid transport and cholesterol homeostasis. As the only natural source of this protein, human blood cannot provide large-scale ApoE3 for research and applications. Therefore, in our study, a Pichia pastoris expression system was first used to obtain a high-level expression of secreted, recombinant human ApoE3 (rhApoE3).The full-length sequence encoding ApoE3, gained by RT-PCR, was inserted into the pPICZαC vector and transformed into P. pastoris strain X33, and then the high expression transformants with zeocin resistance were obtained. The growth conditions of the transformant strains were optimized in 50 ml conical tubes including pH and inducing time. After induction with methanol, the expression level of rhApoE3 was 120 mg/L in 80 L fermentor. RhApoE3 was purified more than 94% purity using SP Sepharose ion exchange chromatography and source™ 30RPC. A preliminary biochemical characterization of purified rhApoE3 was performed by analyzing the ability of inhibiting PDGF-induced proliferation of rat coronary artery smooth muscle cells (SMCs), and the results demonstrated that the function of purified rhApoE3 was similar to natural human ApoE3.     

High-level secretion of biologically active aprotinin from the yeastPichia pastoris

Summary A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site.Pichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion.P. pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.     

Pilot-scale fermentation, purification, and characterization of recombinant human Oncostatin M in Pichia pastoris

Oncostatin M (OSM) is a multifunctional cellular regulator that belongs to the IL-6 subfamily and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. In order to achieve the higher level yield of recombinant human Oncostatin M (rhOSM), we determined the optimal pH condition of rhOSM expressed in the methylotrophic yeast Pichia pastoris X-33 and carried out the fermentation culture of rhOSM in 80 L fermentor in a fed-batch mode. SDS–PAGE and Western blotting assays demonstrated that rhOSM was successfully expressed and secreted into the culture medium with an apparent molecular weight of 28 kDa. N-terminals were correctly processed through amino-terminal sequencing. The maximum yield of rhOSM was 280 mg/L. rhOSM was purified by phenyl Sepharose hydrophobic interaction chromatography and SP Sepharose Fast Flow cation exchange chromatography, which resulted in a final yield of purified rhOSM of 6.94 g with a recovery of 62% and a purity of 95%. The purified rhOSM had a specific growth inhibition activity of 6.26 × 10⁴ RU/μg, which was commensurate with typical values (6.2 × 10⁴ RU/μg) obtained with standard hOSM.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Application of proteoglycan extracted from the nasal cartilage of salmon heads for ex vivo expansion of hematopoietic progenitor cells derived from human umbilical cord blood

Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis.     

Expression, purification, and functional characterization of recombinant human interleukin-7

Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7's effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler purification steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple purification strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the purified rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a specific activity comparable to commercial sources. These results suggest that purification of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL-7 for clinical applications as well as basic researches.     

Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris

Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.     

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 g (wet weight) of cells (0.5 L of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molecular mass of 59.228 ± 0.064 kDa, which agrees with the calculated molecular weight of 59.172 kDa for the rMAOB protein sequence assuming one mole of covalent FAD per mole of the enzyme. Consistent with the MALDI-MS data, purified rMAOB shows a single band near 60 kDa in Coomassie-stained SDS–PAGE gel as well as on Western blot analyses performed using antisera raised against human MAOA and BSA-conjugated FAD. A partial amino acid sequence of the purified protein is confirmed to be that of the wild type rMAOB by in-gel trypsin digestion and MALDI-TOF-MS analyses of the liberated peptide fragments. Steady state kinetic data show that purified rMAOB exhibits a K_m(amine) of 176 ± 15 μM and a k_cat of 497 ± 83 min⁻¹ for benzylamine oxidation, and a K_m(O₂) of 170 ± 10 μM. Kinetic parameters obtained for purified rMAOB are compared with those reported earlier for recombinant human liver MAOB expressed in P. pastoris.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Cloning,large-scale production,and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164)

Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation (～124 mg/L) and purified by heparin affinity chromatography. SDS–PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48 kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3 days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

High-level expression of a biologically active staphylokinase in Pichia pastoris

Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310?mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20–37°C and pH ranges of 6.8–9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042?U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720?µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6?kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.