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  1. Fulltext Effect of codon optimisation on the production of recombinant fish growth hormone in Pichia pastoris
    Rothan HA, Huy TS, Mohamed Z
    ScientificWorldJournal, 2014;2014:514835.
    PMID: 25147851 DOI: 10.1155/2014/514835
    This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
    Matched MeSH terms: Pichia/metabolism
  2. Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris
    Lau YL, Fong MY, Idris MM, Ching XT
    Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):10-6.
    PMID: 23082548
    Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.
    Matched MeSH terms: Pichia/metabolism*
  3. Fulltext Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis
    Chang PY, Fong MY, Nissapatorn V, Lau YL
    Am J Trop Med Hyg, 2011 Sep;85(3):485-9.
    PMID: 21896809 DOI: 10.4269/ajtmh.2011.11-0351
    Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).
    Matched MeSH terms: Pichia/metabolism*
  4. Fulltext Optimization of physical conditions for the production of thermostable T1 lipase in Pichia guilliermondii strain SO using response surface methodology
    Abu ML, Nooh HM, Oslan SN, Salleh AB
    BMC Biotechnol, 2017 Nov 10;17(1):78.
    PMID: 29126403 DOI: 10.1186/s12896-017-0397-7
    BACKGROUND: Pichia guilliermondii was found capable of expressing the recombinant thermostable lipase without methanol under the control of methanol dependent alcohol oxidase 1 promoter (AOXp 1). In this study, statistical approaches were employed for the screening and optimisation of physical conditions for T1 lipase production in P. guilliermondii.

    RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.

    CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.

    Matched MeSH terms: Pichia/metabolism*
  5. The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system
    Abu ML, Mohammad R, Oslan SN, Salleh AB
    Prep Biochem Biotechnol, 2021;51(4):350-360.
    PMID: 32940138 DOI: 10.1080/10826068.2020.1818256
    A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
    Matched MeSH terms: Pichia/metabolism*
  6. Fulltext Characterisation of a truncated Toxoplasma gondii surface antigen 2 (SAG2) secreted by the methylotrophic yeast Pichia pastoris
    Lau YL, Shamilah H, Fong MY
    Trop Biomed, 2006 Dec;23(2):186-93.
    PMID: 17322821 MyJurnal
    A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.
    Matched MeSH terms: Pichia/metabolism*
  7. Fulltext Production of recombinant human epidermal growth factor in Pichia pastoris
    Eissazadeh S, Moeini H, Dezfouli MG, Heidary S, Nelofer R, Abdullah MP
    Braz J Microbiol, 2017 Apr-Jun;48(2):286-293.
    PMID: 27998673 DOI: 10.1016/j.bjm.2016.10.017
    This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27μg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
    Matched MeSH terms: Pichia/metabolism*
  8. Fulltext A comparative study on the expression, purification and functional characterization of human adiponectin in Pichia pastoris and Escherichia coli
    Rothan HA, Teh SH, Haron K, Mohamed Z
    Int J Mol Sci, 2012;13(3):3549-62.
    PMID: 22489167 DOI: 10.3390/ijms13033549
    Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.
    Matched MeSH terms: Pichia/metabolism*
  9. Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents
    Joseph NM, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS
    Biotechnol Prog, 2016 Jul 08;32(4):1038-45.
    PMID: 27088434 DOI: 10.1002/btpr.2279
    The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.
    Matched MeSH terms: Pichia/metabolism*
  10. Fulltext Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents
    Xin J, Wan Mahtar WNA, Siah PC, Miswan N, Khoo BY
    Mol Med Rep, 2019 Jun;19(6):5368-5376.
    PMID: 31059050 DOI: 10.3892/mmr.2019.10201
    Cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. Currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high‑throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMD1168H expressing DNA topoisomerase I (SMD1168H‑TOPOI) in a yeast‑based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMD1168H‑TOPOI as compared with that in SMD1168H. Glutamate also enhanced the growth of the SMD1168H; however, the growth effect was not enhanced in SMD1168H‑TOPOI treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H‑TOPOI. The findings of the current study indicated that SMD1168H‑TOPOI has similar characteristics to MDA‑MB‑231 cells; therefore, it can be used in a yeast‑based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells.
    Matched MeSH terms: Pichia/metabolism
  11. Fulltext Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology
    Tam YJ, Allaudin ZN, Lila MA, Bahaman AR, Tan JS, Rezaei MA
    BMC Biotechnol, 2012 Oct 05;12:70.
    PMID: 23039947 DOI: 10.1186/1472-6750-12-70
    BACKGROUND: Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.

    RESULTS: The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.

    CONCLUSIONS: The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

    Matched MeSH terms: Pichia/metabolism
  12. Fulltext Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12
    Ramli AN, Mahadi NM, Rabu A, Murad AM, Bakar FD, Illias RM
    Microb Cell Fact, 2011;10:94.
    PMID: 22050784 DOI: 10.1186/1475-2859-10-94
    Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.
    Matched MeSH terms: Pichia/metabolism
  13. Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris
    Fong MY, Lau YL
    Parasitol Res, 2004 Jan;92(2):173-6.
    PMID: 14655048
    A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
    Matched MeSH terms: Pichia/metabolism*
  14. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment
    Abd Wahid MA, Megat Mohd Noor MJ, Goto M, Sugiura N, Othman N, Zakaria Z, et al.
    Biosci Biotechnol Biochem, 2017 Aug;81(8):1642-1649.
    PMID: 28585494 DOI: 10.1080/09168451.2017.1329617
    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.
    Matched MeSH terms: Pichia/metabolism
  15. Fulltext Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris
    Karim KM, Husaini A, Hossain MA, Sing NN, Mohd Sinang F, Hussain MH, et al.
    Biomed Res Int, 2016;2016:5962028.
    PMID: 27504454 DOI: 10.1155/2016/5962028
    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
    Matched MeSH terms: Pichia/metabolism*
  16. Fulltext Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris
    Abubakar MB, Aini I, Omar AR, Hair-Bejo M
    J Biomed Biotechnol, 2011;2011:414198.
    PMID: 21541235 DOI: 10.1155/2011/414198
    Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).
    Matched MeSH terms: Pichia/metabolism*
  17. Fulltext Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1
    Jambari NN, Liddell S, Martinez-Pomares L, Alcocer MJC
    PLoS One, 2021;16(4):e0249876.
    PMID: 33914740 DOI: 10.1371/journal.pone.0249876
    Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.
    Matched MeSH terms: Pichia/metabolism
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