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.     

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.     

Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus.

The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media. The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.     

重组人IL-4大肠杆菌表达与纯化

根据大肠杆菌密码子偏爱性优化并合成人白细胞介素4基因,以pET30a( )为载体构建了重组表达质粒pET30a( )/rhIL-4,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,诱导表达并超声破菌检测重组蛋白的表达形式。采用5L发酵罐培养工程菌,发酵液OD600为0.6时诱导3.5h收集菌体,检测目的蛋白的表达量。收集的菌体经压榨破菌获得包涵体,通过包涵体变性、层析、透析复性等方法对rhIL-4进行纯化。采用人红细胞白血病细胞(TF-1)测定纯化的rhIL-4的生物活性。测序表明目的基因已插入载体pET30a( )中,重组蛋白以包涵体形式表达,单位体积重组蛋白的表达量达200mg/L发酵液,建立了对包涵体形式表达的rhIL-4纯化方法,最终得率为40mg/L发酵液,纯度大于98%,回收率为20%以上。免疫印迹法检测诱导表达的重组蛋白和纯化的蛋白为IL-4,N端氨基酸序列测定结果与理论相符,生物活性检测纯化的蛋白比活性达2.5×106AU/mg。这为rhIL-4进一步产业化研究建立了基础。     

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 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.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris

Interleukin-2 (IL-2) plays important roles in variety of immune functions. Recombinant IL-2 has become an important therapeutic protein for therapy of melanoma and renal cell carcinoma. Previously, it was proved that the therapeutic efficacy of rIL-2 expressed in Saccharomyces cerevisiae was improved by prolonging its in vivo half-life through genetic fusion with albumin. In this study, a fusion protein composed of hIL-2 genetically fused to HSA was expressed as a secretory protein under AOX1 (alcohol oxidase 1) promoter in Pichia pastoris. An effective strategy was established to express rhIL-2-HSA fusion protein in 5L scale and the optimal purification procedure was investigated. The purity of rhIL-2-HSA in final product was about 95%. The purified rhIL-2-HSA fusion protein could be recognized by both anti-hIL-2 and anti-human serum albumin monoclonal antibody. Bioactivity analysis showed that the purified rhIL-2-HSA fusion protein displayed high level activity on proliferation in IL-2 dependent manner in CTLL2-cells. rhIL-2-HSA fusion protein also showed a extended half-life in plasma compared with IL-2 when tested in a BALB/c mouse model. This study provides an alternative strategy for large-scale production of bioactive rhIL-2-HSA fusion protein using P. pastoris as an expression host.     

Expression of recombinant human interleukin 6 ( rhIL6) in Saccharomyces cerevisiae by the modified phosphoglycerate kinase and chelatin promoter

Using the modified yeast expression vectors that contained phosphoglycerate kinase or chelatin promoter with the adenine (A) base at –3 position from the ATG coding sequences and the leu-2 gene, recombinant human inter-leukin-6 ( rhIL-6) was produced as a b-galactosidase ( lacZ) fusion protein in Saccharomyces cerevisiae. Expression level of the IL-6-lacZ was 12 times higher than that from the unmodified wild type plasmids and IL-6 activity was 9–9.8 x 10 5 unit/mg yeast total protein. Thus these modified yeast vectors are useful for high expression of foreign genes in yeast     

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.     

High-level soluble expression, purification, and functional characterization of the recombinant human leukemia inhibitory factor: a potential general strategy for the recombinant expression of cytokines consisting of four α-helices in a bundle

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95? purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four α-helices in a bundle for basic research, as well as clinical applications.     

Cloning, expression, purification and functional characterization of recombinant human PSP94

Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS-PAGE analysis and a peak with a molecular mass (M(r)) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, in vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.     

High level expression of active recombinant human interleukin-3 in Pichia pastoris

Interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. A DNA fragment containing the mature human IL-3 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-3 production were identified, secreting as much as 26 mg/L rhIL-3 after 4 days of induction by methanol in flask. The rhIL-3 was purified by Ni⁺-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-3 preparation at about 21 mg/L. Mass spectrometry and MALDI-TOF-TOF analysis of the purified rIL-3 showed molecular weights of 18995.694 Da and 22317.469 Da, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-3 proteins was confirmed by its ability to support ba/f3 cells proliferation and activate the ERK signaling pathways. The results demonstrate that the experimental procedure we have developed can produce a large amount of active recombinant human IL-3 from P. pastoris.     

Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells

Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical 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.     

Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).     

A novel melittin-MhIL-2 fusion protein inhibits the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth

In the current study, we produced a novel fusion protein (melittin-mutant human interleukin 2, melittin-MhIL-2) comprising a mutant human interleukin 2 (Arg88/Ala125) genetically linked to melittin. The plasmid pET15b-melittin-MhIL-2 (Arg88/Ala125) was transformed into E. coli for protein expression. The expressed melittin-MhIL-2 protein was purified using a series of purification steps. The interleukin 2 (IL-2) activity of melittin-MhIL-2 fusion protein was compared with recombinant human interleukin 2 (rhIL-2) for its ability to induce CTLL-2 proliferation. Moreover, the fusion protein directly inhibits the growth of human ovarian cancer SKOV₃ cells in vitro. In an in vivo initial experiment, the fusion protein inhibited tumor growth in ovarian cancer mice. In conclusion, we generated a novel melittin-MhIL-2 fusion protein that retained functional activity of IL-2 and melittin and inhibited tumor growth in vivo.     

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.     

Expression and Purification of Human Membrane Progestin Receptor α (mPRα)

Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3–1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.