Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.     

Expression,purification, and characterization of recombinant human neurturin secreted from the yeast Pichia pastoris

Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the alpha-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.     

Large-scale production,purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris

The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris. To optimise yields of PHA-E, transformants of P. pastoris were selected for high-level production of the recombinant protein. A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported. PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography. The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa. Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein. The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E. The data presented here suggest that, using P. pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.     

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9)IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha2b for both research and industrial purpose.     

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Key words proinsulin; C-peptide; Pichia pastoris     

Expression and functional characterization of a recombinant targeted toxin with an uPA cleavable linker in Pichia pastoris

A recombinant targeted toxin (Disintegrin-Conj-Mel) was developed that contained a disintegrin connected to cytotoxic melittin by a urokinase plasminogen activator (uPA)-cleavable linker. This recombinant targeted toxin was designed to target tumor cells expressing integrin αvβ3. The fusion gene was expressed under the control of the promoter AOX1 in Pichia pastoris. Electrophoresis by SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain, demonstrated that an approximately 13 kDa fusion protein was secreted into the culture medium. The molecular weight was that calculated from the predicted amino acid sequence. After optimizing the growth and expression conditions of the transformant strain, about 160 mg/L of the recombinant protein was achieved. The recombinant protein was purified to more than 95% purity by SP Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The hemolysis bioactivity test revealed that the fusion had no hemolytic activity or cytotoxicity against uPA non-expressing 293 cells, but exerted dose-dependent inhibition on uPA-expressing A549 cell proliferation.     

Expression, purification, and characterization of recombinant Metarhizium anisopliae acid trehalase in Pichia pastoris

The mature peptide of Metarhizium anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant ATM1 (reATM1) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1 in the culture supernatant reached the peak, 5.35 U/mg. Enzyme with a histidine sequence appended to the C terminus was still active and was purified using metal-chelate affinity chromatography. The yield of purified reATM1 was 2.5 mg from 1L supernatant. The purified reATM1 exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. The optimum temperature and pH of reATM1 were 30 degrees C and 6.0, respectively, and the K(m) and V(max) values for reATM1 were 2.6 mM and 0.305 mmol/min/mg, respectively. Studies showed that the enzymatic properties of reATM1 were similar to those of the native ATM1.     

Overexpression, purification and characterization of the Trichoderma atroviride endochitinase, Ech42, in Pichia pastoris

The endochitinase gene ech42 from Trichoderma atroviride was cloned and expressed in Pichia pastoris using a constitutive expression system. Over 98% of the recombinant protein was secreted into the culture medium as glycoprotein. A high endochitinase concentration, 186 mg/L with a specific enzyme activity of 14,128 Umg(-1) was produced. The optimal enzyme kinetic parameters for the recombinant protein were identical to those reported for the enzyme isolated from T. atroviride. The recombinant endochitinase possesses suitable features for biotechnological applications, such as activity at acidic pH and thermostability.     

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.     

重组虎纹捕鸟蛛毒素—I在巴氏毕赤酵母中的表达及纯化

虎纹捕鸟蛛毒素I是从虎纹捕鸟蛛粗毒中分离纯化,具有镇痛活性的肽类神经毒素。对巴氏毕赤酵母生产的重组HWTX－I进行多步纯化,首先将分泌到培养上清的rHWTX-I进行90％饱和度的（NH4）2SO4沉淀,再用截留分子量3kD的滤膜脱盐,再用CM阳离子交换层析分离,最后用C18反相层析脱盐纯化,真空干燥后得到的rHWTX-I经Tricine SDS-PAGE,质谱鉴定,氨基酸组成分析,N－端序列测定后活性鉴定,证明已获得高纯度的重组HWTX－I,摇瓶表达量约为80mg/L,约占总分泌量的23．6％,并对摇瓶发酵条件进行了优化,为利用基因工程方法生产HWTX－I的规模化生产及临床应用提供了证据。     

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.     

Expression of kringle 5 domain of human plasminogen in Pichia pastoris

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.     

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.     

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.     

High-level expression and purification of recombinant human catalase in Pichia pastoris

Catalase is one of the antioxidant enzymes and is involved in many pathophysiologic processes and human diseases. This study focused on high-level expression and purification of recombinant catalase in Pichia pastoris. The cDNA encoding catalase was cloned by RT-PCR from Fetal liver of Homo sapiens. After PCR and construction of expression vector pPIC9K-CAT, human catalase was expressed highly in P. pastoris yeast SMD1168 and secreted into the culture medium. The secreted catalase was purified to a purity of 95% by ammonium sulfate fractionation, anionic exchange-chromatography, and Macro-prep Ceramic Hydroxyapatite with a overall yield of 60%. This study provides a new method for large-scale expression and purification of recombinant protein catalase.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

人Tumstatin在毕赤酵母中的表达和活性分析

利用PCR技术从重组质粒pET-3c-tum中扩增人tumstatin的cDNA片段,连入pPICZαA酵母表达载体,获得的重组质粒pPICZα-tum电激法转化毕赤酵母GS115。经表型鉴定、诱导表达筛选,得到可分泌表达人tumstatin的重组酵母转化子,表达蛋白质的相对分子量约30kD,表达量约25mg/L。表达上清经超滤浓缩和离子交换法初步纯化,所得产物具有免疫活性,能够抑制内皮细胞增殖,诱导其发生细胞凋亡,并能抑制鸡胚尿囊膜血管生成。