Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.     

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.     

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.     

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.     

Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris

As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.     

Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli

Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli.     

High-level expression and purification of Escherichia coli oligopeptidase B

Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.     

Expression and purification of a cold-adapted group III trypsin in Escherichia coli

The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.     

Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts

The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more efficient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal alpha factor signal sequence or at the C-terminus failed to assist in purification by immobilized metal-ion affinity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its purification by IMAC, following hydrophobic interaction chromatography. The purified recombinant dalcochinase was apparently composed of differently post-translationally modified forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure-function relationships in this enzyme.     

Recombinant Bacterial Expression of the Lysozyme from the Tobacco-Hornworm <Emphasis Type="Italic">Manduca sexta</Emphasis> with Activity at Low Temperatures

A gene coding for lysozyme from the insect Manduca sexta (Ms-lyz) was expressed in Escherichia coli. The protein was produced as an insoluble cytoplasmic inclusion body which was denatured in 8 M guanidine-HCl, renatured and purified by affinity and ion-exchange chromatography. The N-terminal sequence and the activity of the recombinant protein against Micrococcus luteus confirmed that correct expression had occurred. When Ms-lyz activity was compared to hen egg white lysozyme, the insect lysozyme was active at lower temperatures. These results demonstrate the feasibility of producing a disulfide-bonded lysozyme enzyme in bacteria and suggest that the insect Ms-lyz is an interesting system for further development of an antibacterial functional at low temperatures.     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

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.     

High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp)

Phospholipase D (PLD) is one of the main enzymes involved in signal transduction, vesicle trafficking and membrane metabolism processes. Here we describe the heterologous high-yield expression in the yeast Pichia pastoris, one-step purification and characterization of catalytically active PLDalpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDalpha is recognized by a polyclonal antibody raised against native soybean PLDalpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDalpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1L of yeast culture medium, about 8 mg of pure recombinant PLDalpha was obtained and the specific activity measured on phosphatidylcholine was 27 micromol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDalpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDalpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.     

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.     

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.