Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH₄OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins. Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

High production of laccase B from <Emphasis Type="Italic">Trametes</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO₄ and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

High-level expression of non-glycosylated and active staphylokinase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.     

Recent advances on the GAP promoter derived expression system of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review. Supported by the National High-tech R&D Program (863 program) (No.2007AA021307).     

Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.     

Cloning and expression of human lectin-like oxidized low density lipoprotein receptor-1 in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

Lectin-like oxidatively-modified LDL receptor-1 (LOX-1) is a major receptor for oxidized low-density lipoprotein (oxLDL) in aortic endothelial cells. Human LOX-1 (hLOX-1) gene (cDNA) was cloned from the monocytic leukemic cell line THP-1 and expressed in Pichia pastoris. The recombinant protein (rhLOX-1) was purified by his-tag affinity chromatography. Preliminary identification was performed by Western blot analysis and a ligand-receptor binding assay showed that the protein had specific oxLDL-binding activity.Revisions requested 21 September 2004; Revisions received 10 November 2004     

Production of bifunctional single-chain antibody-based fusion proteins in <Emphasis Type="Italic">Pichia </Emphasis><Emphasis Type="Italic">pastoris</Emphasis> supernatants

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.     

Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in anti-tumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis to a degree of about 32.4%. Taken together, the FIP gene from Changbai G. lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l⁻¹). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimized expression of an acid xylanase from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its biochemical characterization

The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His⁺ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA and Co²⁺ ion and strongly inhibited by Mn²⁺, Li⁺ and Ag⁺ ions. The K _m and V _max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Expression,purification and characterization of anti-BAFF antibody secreted from the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a ‘yeastern blotting’ method. The scFv-Fc antibody was purified and 56 mg was obtained from 1 l of culture supernatant. It retained high binding activity to both soluble BAFF and membrane-bound BAFF.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

High-yield purification and characterization of recombinant human leukotactin-1 in<Emphasis Type="Italic">Pichia pastoris</Emphasis>

The human chemokine, the short version of leukotactin-1 (shLkn-1; molecular weight =7.2 kD and 66 amino acids), was expressed and secreted into a culture medium using the methylotrophic yeast,Pichia pastoris. The recombinant shLkn-1 was purified from the culture supernatant using a simple two-step procedure consisting of cation exchange and reverse phase chromatography (RPC), in which shLkn-1 was highly purified (99.5%) with a high recovery yield of 82.7%. The C-terminal truncated derivative of shLkn-1 was found in the supernatant and was separated by RPC. The physicochemical properties of the purified shLkn-1 were verified to be the same as expected. The biological activity of the purified recombinant shLkn-1 was also quantified using a chemotaxis assay. It was observed that the recombinant shLkn-1 had the maximum migration activity at a concentration of 10 nM, as potent as MIP-1α.     

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N ⁴-(N-acetyl-β-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannose-type structures. Subfractionations were carried out via normal phase (Lichrosorb-NH₂) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz ¹H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man₈GlcNAc₂ to Man₁₁GlcNAc₂ including molecular isomers, could be established, and structures up to Man₁₅GlcNAc₂ were indicated. Phosphate-containing oligosaccharides ranged from Man₉ PGlcNAc₂ to Man₁₃ PGlcNAc₂. Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.     

Changing the N-terminal sequence protects recombinant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> circumsporozoite protein from degradation in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) _n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.