Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.     

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.     

Expression and characterization of a synthetic protein C activator in Pichia pastoris

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1). In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.     

Overexpression of a small medicinal peptide from ginseng in the yeast Pichia pastoris

A medicinal peptide, Gsp, which was initially extracted from the traditional medicinal herb ginseng, has potential use as a drug against diabetes. Gsp is a low molecular weight protein that we have secreted in a recombinant form from the yeast Pichia pastoris. A DNA fragment encoding four copies of the Gsp protein each separated by a basic amino acid was synthesized and inserted into the P. pastoris expression vector plasmid pPIC9. After electroporation of the resulting vector, pPIC9-Gsp, into the yeast, transformants were selected. Recombinant pre-Gsp secreted from P. pastoris had a molecular weight of 5.9 kDa and mature recombinant Gsp had a primary structure indistinguishable from native Gsp. After optimization of the culturing process, the yield of pre-Gsp reached 800 mg/L in the clarified broth. A continuous batch fermentation process was developed that allowed the same population of cells to be reutilized five times without loss of expression level. This continuous culturing process resulted in a substantial saving of both time and cost in pharmaceutical production and should be applicable to the production of other recombinant proteins in P. pastoris.     

Molecular cloning, expression and purification of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma

A cDNA encoding LAAO from the Malayan pit viper (Calloselasma rhodostoma) was cloned into an expression vector of the methylotropic yeast Pichia pastoris. The LAAO open reading frame was inserted after the alpha-MF-signal sequence. Upon induction soluble and active LAAO is produced and exported into the culture supernatant at a concentration of up to 0.4 mg/L. Recombinant LAAO was purified from this by ion exchange and molecular sieve chromatography to yield apparently homogeneous protein in quantities of approximately 0.25 mg/L growth medium. Expressed LAAO exhibits the same electrophoretic mobility as native LAAO (62 kDa) and exhibits approximately the same extent of glycosylation as authentic LAAO from snake venom. Catalytic properties and substrate specificity of recombinant LAAO are similar to those of native enzyme.     

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.     

High-level expression of Aliciclobacillus acidocaldarius thioredoxin in Pichia pastoris and Bacillus subtilis

Thioredoxins are ubiquitous proteins which catalyze the reduction of disulfide bridges on target proteins and are involved in many cellular reactions. In a previous work, a thioredoxin from the thermophilic organism Aliciclobacillus acidocaldarius (Alitrx) was purified, characterized, and its gene expressed in Escherichia coli. In order to produce larger quantities of Alitrx, the protein has been expressed in the methylotrophic yeast Pichia pastoris and in the gram positive bacteria Bacillus subtilis. The growth conditions of strains showing high-level expression of Alitrx were optimized for both systems in shake-flask cultures. Active proteins were secreted in the culture media at a level of approximately 0.9 and 0.5 g/l, respectively, for P. pastoris and B. subtilis. The proteins were purified almost to homogeneity by a thermal precipitation procedure, with a 90-fold and 50-fold higher total yield with respect to that obtained with the same protein expressed in E. coli. The results indicate that either of these two systems could be utilized as a host for large-scale production of recombinant Alitrx.     

Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.     

Production and characterization of recombinant guamerin, an elastase-specific inhibitor, in the methylotrophic yeast Pichia pastoris

The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography. Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P. pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L. The recombinant guamerin was highly purified (>98%) with a recovery yield of 68%. Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein. The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase. Furthermore, the recombinant guamerin did not produce a humoral immune response in mice.     

Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.     

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.     

Characterization of Trichoderma reesei endoglucanase ii expressed heterologously in Pichia pastoris for better biofinishing and biostoning

The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.     

Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal OD600 of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.     

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.     

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.     

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris

Aims:  To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes.
Methods and Results:  A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62·8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus . Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators.
Conclusions:  Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators.
Significance and Impact of the Study:  This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris

Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of homogeneously pure active PAP without batch-to-batch variation from its natural resource. Here, we report the expression of mature PAP (residues 23 to 284) with a C-terminal hexahistidine tag in the methylotrophic yeast Pichia pastoris, as a secreted soluble protein. The final yield of the secreted PAP is greater than 10 mg/L culture in shaker flasks. The secreted recombinant protein is not toxic to the yeast cells and has an apparent molecular mass of 33-kDa on SDS-PAGE gels. The in vitro enzymatic activity and cellular anti-HIV activity of recombinant PAP were of the same magnitude as those of the native PAP purified from P. americana. To our knowledge, this is the first large-scale expression and purification of soluble and biologically active recombinant mature PAP from yeast.