AOX1 Gene Promoter Research Articles

Effect of Methionine on Gene Expression in Komagataella phaffii Cells.

Komagataella phaffii yeast plays a prominent role in modern biotechnology as a recombinant protein producer. For efficient use of this yeast, it is essential to study the effects of different media components on its growth and gene expression. We investigated the effect of methionine on gene expression in K. phaffii cells using RNA-seq analysis. Several gene groups exhibited altered expression when K. phaffii cells were cultured in a medium with methanol and methionine, compared to a medium without this amino acid. Methionine primarily affects the expression of genes involved in its biosynthesis, fatty acid metabolism, and methanol utilization. The AOX1 gene promoter, which is widely used for heterologous expression in K. phaffii, is downregulated in methionine-containing media. Despite great progress in the development of K. phaffii strain engineering techniques, a sensitive adjustment of cultivation conditions is required to achieve a high yield of the target product. The revealed effect of methionine on K. phaffii gene expression is important for optimizing media recipes and cultivation strategies aimed at maximizing the efficiency of recombinant product synthesis.

Prospects of biosimilar recombinant human growth hormone manufacture in the Republic of Moldova

In collaboration of SUMPh „N.Testemițanu”, the pharmaceutical company Balkan Pharmaceuticals and ICGEB the technology transfer during scale-up from the laboratory phase to the pilot phase of the processes of creating the working cell bank and growing Pichia pastoris strains in the Erlenmeyer flask were carried out. Pichia pastoris was selected for recombinant growth hormone as an expression system, which has many advantages over other organisms, including protein folding, post-translational changes in protein, and easy handling. The GH1 altered recombinant human somatropin gene was introduced into the genome of Pichia pastoris culture using restriction near the AOX1 gene promoter as a vector serving the pPIC9K plasmid. The GH1 gene was activated by the AOX1 gene promoter which can be easily induced by the presence of methanol in the culture medium. Subsequently, the „Prepro-alpha Factor Leader” sequence from Saccharomyces cerevisiae was added to the modified sequence so that the obtained somatropin could be secreted into the culture medium. In the technological transfer process the influence of the chosen technological processes on some parameters of the obtained product were examined, Technological Regulations were developed for short and long term storage processes of Pichia pastoris cells and for the process of inoculum preparation, inoculation and growth of P. pastoris strains with recombinant human growth hormone in the Erlenmeyer flask. Standard Operating Procedures were developed for the processes of creating the working cell bank, determining the in-vitro biological activity of the recombinant human growth hormone preparation, with subsequent implementation in the process of producing rhGH biosimilar at CS Balkan Pharmaceuticals Ltd.

Comparasion of the effectiveness of anchor proteins ScAGα1p, KpCW51p, KpCW61p for surface display in yeast <i>Komagataella phaffii</i>

BACKGROUND: Yeast display is an effective technology for exposure target proteins to the cell surface by fusing them with cell wall proteins. This technique, among other things, makes it possible to obtain vaccine preparations based on yeast by exposing antigen proteins on their cell surface. Finding and selecting proteins that allow effective exposure of target proteins on the surface of yeast cells is an urgent task.
 AIM: The aim of this work was to evaluate the efficiency of cell wall proteins ScAG1p, KpCW51p, KpCW61p for displaying the reporter protein on the Komagataella phaffii cell surface, including the study of several variants of the ScAG1 gene coding sequence.
 MATERIALS AND METHODS: The studied gene sequences were cloned under the control of the AOX1 gene promoter in the same reading frame as the eGFP reporter protein gene and integrated into the genome of the K. Phaffii yeast strain X-33.
 RESULTS: Cytoimmunochemical analysis and confocal microscopy of strains displaying the eGFP protein on their surface under conditions of induction of the AOX1 gene promoter made it possible to identify the most effective anchor protein. The best efficiency was demonstrated for the sequence of the ScAG1 gene without the native 3' non-coding region.
 CONCLUSIONS: The plasmids obtained in the work will make it possible to obtain a yeast strain K. phaffii that effectively exposure proteins, including antigens, on its surface, which can be used as a vaccine preparation.

Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris

BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.
 AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.
 MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).
 RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.
 CONCLUSIONS: Thus, the obtained strains of P. pastoris producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.

Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii.

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)


 
 Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.
 
 
 
 

Influence of PDI gene overexpression on heterological proteins production in yeast Pichia pastoris

Summary: Background. The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins. Overexpression of assistant genes, coding proteins involved in secretion, is one of approaches to improve the production of target protein. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study was to evaluate the effect of Pdi overproduction on recombinant interferons (human interferon-alfa16 and chicken interferon-gamma) production.
 Materials and Methods. PpPDI gene was cloned under the control of the AOX1 gene promoter in plasmid pPICZA. Primers for AJ302014.1 nucleotide sequence of NCBI data base were used for PpPDI gene cloning. The chromosomal DNA of the GS115 strain was used as a template. To generate strains with PpPDI gene overexpression we used a previously obtained strains producing human interferon-alfa16 and chicken interferon-gamma. Yeast transformation was performed by electroporation. Cultivation was performed using single and two-stage strategies in standard media containing methanol as the sole carbon source to induce the AOX1 gene promoter.
 Results. We obtained interferon-producing strains with PpPDI gene overexpression. Over-expression of the PpPDI gene in yeast P. pastoris increases the production of interferon-alfa16, a protein containing disulfide bonds, regardless of the mode of cultivation. Effect of PpPDI gene over-expression on the production of interferon-gamma the protein without disulfide bonds, depends on cultivation mode.
 Conclusion. PpPDI gene overexpression can be used to enhance the production of interferons and other proteins that contain disulfide bonds. Effect of PpPDI gene overexpression on recombinant proteins without disulfide bonds may depend on cultivation procedure.

Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris.

To confirm the treating effectiveness of midkine as an articular protective agent, mouse midkine (mMK) was produced for the pre-clinic long-term studies in mice. The protein was expressed under the control of the AOX1 gene promoter in Pichia pastoris, X-33 strain, and secreted into fermentation broth through high-density fermentation. Approximately 380 mg mMK, containing authentic and truncated forms, was secreted into 1 liter induction medium and 280 mg mMK was obtained after one-step purification on a 50 ml SP Sepharose Fast Flow column. The purified protein was characterized and identified to be the mature, authentic form of mMK. N-terminal five amino acid sequence was determined to be K-K-K-E-K. SDS-PAGE analysis indicated that the molecular weight of the product was about 13 KDa. The purity of the purified rmMK protein was determined to be 99% by high performance liquid chromatography. The biological activity of final product was verified via migration assay on osteoblast-like UMR-106 cells.

Heterologous interferons synthesis in yeast Pichia pastoris

The HuIFNA16, HuIFNB, and BoIFNG genes encoding human [alpha]16, beta-interferons and bovine gamma-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the "inclusion bodies." The treatment of human beta-interferon with endoglycosidase H showed that protein was expressed in glycosilated and unglycosilated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g −1, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l −1, resulted in a dramatic increase in volumetric yield (85,600 U l −1) and global volumetric productivity (1083 U l −1 h −1).

PEGylated murine Granulocyte–macrophage colony-stimulating factor: Production, purification, and characterization

Granulocyte–macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn’s disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut S strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2–3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.

Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the Mut S strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Characterization of partially truncated human midkine expressed in Pichia pastoris.

Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichiapastoris. Approximately 640 mg of rh-midkine was secreted into one liter of medium of the high cell-density fermentation. The protein processing of the rh-midkine was done efficiently and correctly in P. pastoris, and O-mannosylation was not detected in the purified rh-midkine. However, only about 30% of the purified rh-midkine was intact. The other ones lost 5-12 amino acid residues from the amino-termini, provably by proteolysis. Even the mixture of these truncated midkines could promote CHO cell proliferation as well as the authentic rh-midkine.

Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

Cloning and expression of cDNAs encoding α1,3-fucosyltransferase homologues from Arabidopsis thaliana

The core α1,3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic. We report the identification, isolation and characterisation of the cDNAs of three genes (FucTA, FucTB and FucTC) encoding proteins similar to α1,3-fucosyltransferases in Arabidopsis thaliana. Reverse transcription–polymerase chain reaction was used to amplify the full length coding sequence of FucTA. The FucTA gene, which consists of seven exons, encodes a presumptive protein of 501 amino acids showing an overall sequence identity of 66% to the protein encoded by the recently isolated mung bean Fuc-T C3 cDNA. FucTA was expressed in Pichia pastoris under the control of the AOX1 gene promoter. The soluble enzyme was found to catalyse the same reaction as mung bean core α1,3-fucosyltransferase as judged by analyses of the products by MALDI-TOF and high-performance liquid chromatography. The FucTB cDNA was isolated from a λ-ZAP library, but the clone used an alternative splicing site between the second and third exon resulting in a premature stop codon. The FucTC gene encodes a protein with less than 40% identity to FucTA across 115 amino acids of a total of 401 amino acids and is a member of a new sub-family of plant α1,3/4-fucosyltransferase homologues.

Comparison of Three Signals for Secretory Expression of Recombinant Human Midkine in Pichia pastoris

The secretion signals of Saccharomyces cerevisiae alpha mating factor, human midkine itself, and Pichia pastoris acid phosphatase, were tried for the expression of human midkine under the control of the AOX1 gene promoter in P. pastoris. Approximately 28 mg/l, 1.5 mg/l, and 0.2 mg/l of midkine were secreted by using the a mating factor pre-pro-sequence, the midkine signal sequence, and the phosphatase signal sequence in flask cultures, respectively.

An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis.

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Production of recombinant human midkine in yeast, Pichia pastoris

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/ l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/ l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

Production of Recombinant Human Midkine in Yeast, Pichia pastoris.

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.