• High yield Expression and Purification of A. flavus Uricase Cloned in Pichia pinkTM Expression System
  • Behrouz Mahmoudi ,1 Fatemeh Soleimanifar ,2 Mohammad Ahmadi ,3 Mohammad Parand ,4 Sanaz Mahboudi ,5 Hosein Mahboudi ,6,*
    1. Department of Medical Biotechnology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
    2. Department of Medical Biotechnology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran.
    3. Research and Development Department, Barsam Pharmed Company, Karaj, Iran
    4. Research and Development Department, Persisgen Par Company, Tehran, Iran.
    5. Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, Iran
    6. Department of Medical Biotechnology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran.


  • Introduction: Enzyme-based drugs are part of a very diverse group of protein drugs that catalyze reactions specifically and with minimal interference with biochemical processes (1, 2). Rasburicase, the recombinant form of uricase, is one of the most well-known enzymes in the treatment of various origins of hyperuricemia in humans, which are caused by its evolutionary deletion in the metabolic pathways of uric acid decomposition or complications caused by chemotherapy such as tumor lysis syndrome (3, 4, 5). Its homotetrameric structure in Aspergillus flavus has 301 amino acids in each subunit with a molecular weight of 135 kDa (6). This enzyme was recognized as a suitable substitute for allopurinol in the treatment of hyperuricemia, which has a clear relationship between this condition and tumor lysis syndrome, blood pressure, coronary heart disease, and progressive kidney failure, because the resulting allantoin has ten times more solubility than uric acid. It does not have the harmful effects caused by the accumulation of xanthine and hypoxanthine due to the use of allopurinol (7,8). With the existence of injectable vials for the treatment of hyperuricemia, efforts have been started to test the oral drug enzyme, especially in the cause of renal failures (9). Various prokaryotic and eukaryotic hosts have been used to express rasburicase. One of the first transformations of Aspergillus flavus uricase gene and comparing its expression with the mutant of the same gene was done by Chevalet et al. (10). Initial attempts to clone Aspergillus flavus uricase were made by Legous et al. in E. coli (11). Since then, uricase gene from different sources such as rat, Nilaparvata lugens insect, Arthrobacter globiformis, Bacillus subtilis, Candida utilis, Pseudomonas aeruginosa and Kluyveromyces marxianus has been cloned as a gene construct in different hosts such as E. coli, Saccharomyces cerevisiae and Pichia pastoris (12-19). Among the reasons for the production and use of the recombinant form of uricase (rasburicase) instead of its non-recombinant form under the brand name Uricozyme, is the lower heterogeneity of rasburicase, as well as the 50% increase in specific activity and the removal of host protein and nucleic acid impurities in its production process pointed out (20). Therefore, high-scale expression and maintaining catalytic activity have been among the challenges of producing therapeutic enzymes, which depends on the type of gene expression vector, the host, and the purification strategy used (19). The engineered pichia pink strain not only has the advantages of its mother strain, Pichia pastoris, including protein processing, proper folding, post-translational modifications, and more than a tenfold increase in the expression of heterologous proteins compared to S. cerevisiae, but also due to the gene complementation process ( Complementation) ADE2 causes easy selection of colonies expressing the target gene, compared to the antibiotic resistance method, and on the other hand, due to the knockout of cellular proteases, it reduces their effects and removes the need for heavy protease inhibitors (21, 22). In this research, the synthetic DNA sequence of uricase enzyme was expressed intracellulary after codon optimization using the Ppink-Uox expression vector in the Pichia Pink strain, and after optimizing the expression methods in the fermentor scale, cell lysis and purification, the enzyme was produced with medicinal and industrial potential.
  • Methods: The DNA sequence of urate oxidase (UOX) enzyme was synthesized as PUC57-UOX after codon optimization of Pichia pastoris by GeneScript, USA, and it was used for the transformation of susceptible E. coli Top10 cells. The selected vector for Pichia Pink strain was pPink-HC product of Invitrogen. In order to connect UOX gene to pPink-HC, both vectors were enzymatically digested by EcoR1 and Kpn1 enzymes on 1% agarose gel. Isolation of the UOX gene and the linearized plasmid pPink-HC was performed on agarose gel using the recovery kit of Vivantis company with catalog number 70028C. After making the pPink-UOX expression vector, BTX model ECM630 device with 1800 v, 200 A and 25 ohm parameters was used for electroporation. The enzyme used to linearize the final construct was spe1. After screening the colonies expressing uricase in YPD solid medium, colony PCR was used to confirm the presence of its gene in the recombinant yeast. Using a single colony of transformants, 10 ml of YPD culture medium was inoculated and shaken for 24 hours at 30 degrees at 250 rpm and it was used to inoculate 200 ml of liquid YPD medium until reaching OD600=2-3. The cells were centrifuged at 1500 g for 5 minutes and after discarding the supernatant, the pellete was dissolved in YPD containing 25% glycerol so that the final OD600 reached 100-50 (approximately 5.2-5.1 x 109 cells per milliliter). The cells were aliquot into new vials, frozen by liquid nitrogen and kept at -80°C. Expression of recombinant uricase A single recombinant clone containing UOX gene was inoculated in 10 ml of BMGY medium, then shaken at 30°C and 150 rpm. After centrifugation of the grown cells using Falcon at 1500 g for 5 minutes, the supernatant was discarded and the remaining pellete was dissolved in 10 ml of BMMY medium. After 17±1 hours, 100 microliters of 40% methanol was added to each of the falcons that had been shaken at 150 RPM and at a temperature of 28 ˚C, and they were shaken again with the same conditions as before. After other 17±1 hours, samples were prepared for enzyme activity measurement. After selecting the two colonies with the highest enzyme activity, the cells were induced to express UOX in the presence of different percentages of 0.5, 1 and 3% methanol in BMMY culture medium for 96 hours. To start the fermentation process, after defreezing the cell bank at 37 °C, 400 microliters of it was added to 400 ml of BMGY culture medium and harvested after 17±1 hours incubation and harvested when reaching OD600=3±1 to inoculate the fermantor media. Semi-defined culture medium (SDM) was selected as the fermenter media and the first and second feeds were determined as 400 ml of glycerol (50% w/v) and 1400 ml of methanol (100%). Fermentation was done in a 5-liter Labfors5 model fermenter. The pH of the SDM before inoculation was adjusted to 5.2±0.2 and maintained at this point by adding ammonium hydroxide. After 19±1 hours of inoculation with spike of oxygen, which indicates the complete consumption of glycerol and the beginning of the fedbatch step, glycerol (50% w/v) was added to the culture medium as the first feed at a rate of 20 g/L/h. After the completion of the glycerol feed and the second oxygen spike, 100% methanol as the second feed was started at a rate of 7ml/L/h and reached 26ml/L/h after 48 hours. Finally, after 60 hours and using 1700ml of 100% methanol, it was harvested. 15ml samples were taken every 6 hours to check cell dry weight, OD600, enzyme activity and SDS-PAGE test. Optimal protein expression was performed using 12% SDS-PAGE by adding 25 μl of samples to each well. Bradford's test was also used to determine the protein content. downstream processes Cell lysis Regarding the intracellular expression of recombinant uricase, the following steps were taken to release the enzyme in an active form for every 200 mg of biomass: 200g of harvested biomass in 2L lysis buffer (50mM sodium phosphate, pH=7.4, 1mM PMSF as a protease inhibitor, 1mM EDTA, 5% glycerol) was dissolved at 4°C. The resulting solution was homogenized using a homogenizer (GEA Lab Homogenizer Panda PLUS 2000) in 4 passes at a pressure of 900 bar and treated with PDADMAC 0.05% for 1 hour before centrifugation for 20 minutes at 9000 rpm. Purification of recombinant uricase Ultrafiltration/Diafiltration (UF/DF) In this step, using the lysis buffer and the tangential flow filtration device (TFF 300 kDa) made by Pall Corporation, USA, during 7 cycles, most of the impurities with a molecular weight greater than 300 kDa are removed and the target enzyme is permeated by the 30 kDa filter using the equilibrium buffer (7mM NaH2Po4, 3mM Na2Hpo4, pH=6.5, conductivity=6.5±0.1mS/cm) was concentrated during 7 cycles. Before applying the UF/DF retentate to the column, it was filtered using a 0.22 μm Millipak filter. FPLC To purify tag-free recombinant urate oxidase (uricase) protein, three consecutive columns in GE ÄKTA FPLC™ were used as follows; In the first step of chromatography, the target protein was removed from the DEAE XK Sepharose column in flow through mode. This column (4 x 16 cm, GE Healthcare) was equilibrated using the equilibration buffer used in the TFF device, and in order to maintain the temperature of 4 °C, cold water was circulated through the column and after loading the harvested sample with a resident time of 5 min from the resin was passed. This step was done to remove the host proteins and bind them to the resin, and the recombinant uricase removed from the resin was taken on the CM sepharose column to obtain greater purity. In this column, in addition to the previous equilibrium buffer with pH=6.5 and conductivity=1.35±0.1 mS/cm, Elution buffer with the same formula but with pH=7.86 and conductivity=1.35±0.1 mS/cm and also 2M NaCl as H.salt was used. Finally, the CM column fractions was placed on the Phenyl Sepharose XK 16/20 column with equilibrium buffer (7mM NaH2Po4, 3mM Na2Hpo4, (NH4)2SO4 pH=7.38±1, conductivity=151±1mS/cm), wash buffer (7mM NaH2Po4, 3mM Na2Hpo4, (NH4)2SO4 pH=7.38±1, conductivity=109±1mS/cm), and Elution buffer (7mM NaH2Po4, 3mM Na2Hpo4(NH4)2SO4 pH=7.38±1, conductivity=68±1mS/cm) for final purification. Enzyme specificity analysis, reverse phase chromatography (RPC) and size exclusion chromatography (SEC) were also performed based on the method described in (26). Enzyme activity assay Cell lysis was performed in the expression step in the Erlen and Fermentor scale due to the small volume of the sample using the glass bead method (27). The investigation of uricase enzyme activity was done in all steps after the harvest of the fermenter using UV spectrophotometry optimization (28). 1 ml of the supernatant obtained from the cell lysis step was centrifuged at 14000 rpm for 20 minutes at 4 ˚C. To prepare the standard curve as a positive control, 10 microliters of standard enzyme was added to 290 microliters of boric acid buffer (1 Iu/ml). 20 microliters of the resulting solution was added to 580 microliters of uric acid solution and 13 readings were made using spectrophotometry at a wavelength of 293 nm with 10 second intervals. Enzyme activity was calculated using the following formula: units/ml Enzyme=((∆A293/min⁡〖test-∆A293/min⁡〖blank)〗 〗 (0.6)(DF))/((12.3)(0.02)) 0.6= whole reaction volume per minute 12.3= uric acid extinction coefficient 0.02= volume of enzyme per milliliter
  • Results: Determination of target gene and recombinant expression vectors PUC57-UOX and pPink-UOX. Analysis of uricase, PUC57-UOX and pPink-UOX gene constructs by SDS-PAGE shows that the target gene sequence is correctly placed in the target vectors and between the cutting site of KpnI and Ecor1 enzymes. The uricase gene was under the control of the alcohol oxidase promoter to make the pPink-UOX expression plasmid. To confirm the construction of the recombinant vectors, they were digested with the aforementioned restriction enzymes (Figure 1). Figure (1): Analysis of the stages of cloning, screening and small-scale expression of the recombinant uricase gene. (A) Molecular weight marker (1), undigested vector pPink-HC (2). Linear pPink-HC vector (3), undigested pUC57-UOX (4), pUC57 vector (upper 5) and uricase gene (lower 5), which were both separated by KpnI and Ecor1. (B) Confirmation of uricase gene cloning in pPink-HC after enzymatic digestion and extraction of pPink-HC vector fragments and the target gene (lines 1 and 3 above and below). (C) Linearization of pPink-UOX gene construct with Spe1 enzyme (1), molecular weight marker (2) and non-linear construct pPink-UOX as control (3). (D): Colony PCR results and confirmation of the presence of recombinant uricase gene in the vector of several colonies of 9 Pichia Pink yeast colonies (2 to 9) and molecular weight marker (1). (E) Small-scale expression of recombinant uricase(rUOX) and its SDS-PAGE results in different colonies at three different times 0 (before induction), 24 and 48 hours after cultivation in BMMY medium. Expression of recombinant uricase Finally, after examining the best uricase producing colonies on the Erlen scale, their expression level and enzyme activity were checked in the presence of different percentages of methanol, and one of the colonies was selected for production on the fermenter scale. As the results of SDS-PAGE in Figure (2A) show, the 2D clone on the Erlen scale and with the presence of 0.5% methanol after 96 hours had the higher expression and enzyme activity (0.7 IU/ml) compared to clone 2B and was used for the fermentation process. Both clones showed better performance in the presence of 0.5% methanol, so that the expression and enzyme activity decreased in the presence of 1% and 3% methanol (data not shown). Recombinant uricase comprises 12% of total cellular proteins. Figure (2) SDS-PAGE analysis of recombinant uricase expression in Erlen and Fermentor scales. (A): Expression of two selected colonies in the presence of different percentages of methanol induction after 96 hours. Colony number 2B in the presence of different percentages of methanol 3% (1), methanol 1% (2) and methanol 0.5% (3). Colony number 2D in the presence of different percentages of methanol 3% (4), methanol 1% (5) and methanol 0.5% (6). Urate oxidase standard (7), negative control (8) and molecular marker (9). (B): Recombinant urate oxidase expression in Pichia pink at different hours of Fedbatch fermentor. Molecular markers (1 and 10). 12 h- 60 h of Fermentor fedbatch (3-8 and 12- 14). Negative control (2 and 11). Standard urate oxidase (9 and 15). The conditions for inducing expression in the fermentor-scale were optimized so that there is maximum expression of the enzyme intracellularly. The lysis of cell samples taken from different stages of the fermentor, with increasing hours, showed an increase in expression and also an increase in enzyme activity. The highest yield of intracellular production of recombinant uricase was obtained in semi-defined medium with pH=5.2±0.2, 28±0.5 ˚C, 30% dissolved oxygen, aeration speed 430 rpm, and pressure 0.3±0.1 bar during 60 hours fermentation. About 19±1 hours after inoculation and spike of oxygen in glycerol feed (50% w/v) for 6 hours and following the oxygen spike, 100% methanol was added to the culture medium. Continuous induction with methanol was continued for another 54 hours until its harvest. A separate band of about 34 kDa related to the monomers of recombinant uricase appeared after 18 hours of induction using methanol, which was not visible in the previous hours and became more colorful as the induction hours increased (Figure 2B). Chart (1): Changes of OD600 in different hours of the fedbatch of the fermenter run Fermentor inoculum had OD600=0.273, and at the end of the batch step, this value reached OD600=72 and the cell dry weight reached 30 g/L. Finally, after 60 hours, the OD600 reached 490 and the cell dry weight reached 103 g/L. (Charts 1 and 2). Chart (2): changes in cell dry weight at different hours of the fedbatch of the fermenter run The maximum biomass produced in a fermenter with a culture medium volume of 4 liters is 1500 grams. Considering that glycerol was the first feed of the fedbatch step and this feeding continued for 6 hours and then 100% methanol feeding was started as an inducer of recombinant uricase expression. Therefore, until 12 o'clock in this step, no enzyme activity is observed during sampling. Chart (3): Enzyme activity changes (IU/ml) at different hours of the fedbatch of the fermenter With increasing amount of biomass, with the passage of time, the expression of recombinant uricase also increased, which was accompanied by an increase in enzyme activity (Figure 2B and Figure 3). The enzymatic activity of recombinant urate oxidase reached 2.44 U/ml 6 hours after induction with methanol, and after 60 hours of fermentation it was 14.54 U/ml, which was obtained by glass bead lysis, which showed both expression and very good activity of the enzyme. Purification of recombinant uricase Using a 5-liter fermenter, 1500 grams of biomass was obtained from 4 liters of semi-definite culture medium. All purification steps were performed at 4 ˚C. The target enzyme was purified using three consecutive columns: DEAE sepharose (flow through mode), CM sepharose (capture mode) and Phenyl sepharose (capture mode) after cell lysis and preparation of supernatant (explained in materials and methods). As the SDS-PAGE results of each step show, at the end, a high purity of 99% of the recombinant uricase is obtained. Figure (3) SDS-PAGE analysis of purification steps of recombinant uricase. (A): first step of purification using DEAE sepharose column. Molecular weight marker (1). uricase standard (2). before TFF 300 kDa (3). Permeate 300 kDa (4). Retentate 300 kDa (5). Permeate 30 kDa (6). Retentate 30 kDa (7). Initial (8). Flow through (9). H. salt (10). (B): The second purification step using CM sepharose column. Initial (1). Molecular weight marker (2). Flow through (3). Eluate1(4). Eluate2(5). Eluate 2-1(6). Eluate 2-2(7). Eluate3(8). uricase standard (9). (C): third purification step using Phenyl Sepharose column. Molecular weight marker (1). Initial (2). Wash (3). Eluate (4). H. Salt (5). uricase standard (6). The use of 300 kDa and 30 kDa TFFs, respectively, caused the removal of most of the impurities. The DEAE Sepharose column was run in flow through mode and the recombinant uricase was passed through the column without binding to the resin (Figure 3A). By taking the peak of this step on the CM Sepharose column in capture mode, another major part of impurities is removed (Figure 3B) and eluate peaks with the target enzyme of this column, finally using Phenyl Sepharose resin with a purity of more than 99% of urate oxidase was purified (Figure 3C and Figure 4). The summary of information related to each purification step is given in Table (1). The final yield of recombinant uricase per liter of culture medium was 120 mg. The analysis of the results of RP and SE chromatography shows the purity, hydrophobicity, degraded form and aggregated form of standard enzyme (Figure 5). Figure (4): Chromatogram of the third step of purification using Phenyl Sepharose column The final product was kept for 4 weeks to test the stability of the activity at 4 ˚C (data not shown). Table (1): Purification of recombinant uricase Purification step Volume (ml) Total activity (U) Total protein (mg) Specific activity(IU/mg) Yield Purification (fold) P.pink cell lysate 1000 2500 6596 0.37 100 1 DEAE Sepharose 500 1750 1827 1 70 3 CM Sepharose 308 1190 338 4.37 68 13 Phenyl Sepharose 104 750 31.25 24 63 65 For long-term storage of the enzyme, it was stored through a sterile 0.22 µm filter at -80 ˚C using 20 mM phosphate buffer with 1% mannitol and 1.6% alanine. Figure (5): Size Exclusion Chromatography and Reverse Phase Chromatography analysis results of recombinant uricase. (A): SEC analysis of standard sample. (B): SEC analysis of recombinant uricase. (C): RPC analysis of standard sample. (D): RPC analysis of recombinant uricase.
  • Conclusion: The results of the present research show the successful use of pichia pink yeast in cytosolic expression of recombinant uricase. Creating a new approach in the purification steps of this enzyme led to a purity of over 99%, which is comparable to the biochemical characteristics of the standard enzyme. Choosing Pikia pink as a eukaryotic host and changing the purification approach that can be used in the industry were two unique features in this study, which together enabled the production of recombinant uricase with high expression and optimal preservation of enzyme activity. According to our knowledge up to now, the present study has comprehensively improved the defects in the industrial production of previous studies in terms of host selection, higher expression and the purification approach that can be used in the industry to obtain optimal high enzyme activity.
  • Keywords: Uricase, Pichia pink, FPLC, Rasburicase