Expression and Purification of Receptor Tyrosine Kinase PDGFR? / 微生物学通报

A fusion expression vector pPIC3.5K-PDGFR? was constructed to express recombinant receptor tyrosine kinase PDGFR? and the right Pichia pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of strain GS115, a high yield strain named M3 was screened. The strain M3 was cultured in a 5 L fermentor and His-GFP-PDGFR? fusion protein was purified by Ni2+ chelating affinity chromatography. One distinct peak was obtained after elution with 250 mmol/L imidazole. Fusion protein was proved to be 90.08 kD by western blotting, and have tyrosine kinase activity by ELISA. Results showed that the receptor tyrosine kinase PDGFR? was successfully expressed in P. pastoris and could be used as a target for small molecule selective inhibitors screening.

Construction of Hexose Transporter-like HXT1 Deletion Mutant in Pichia pastoris / 微生物学通报

Glucose was transported by the large number of hexose transporters in yeast cells. There were 18 hexose transporter genes had been identified in Saccharomyces cerevisiae. However,as an excellent expression system,there was no information of these genes had been reported in Pichia pastoris. Based on high homologous recombination efficiency in yeast,we chose G418 resistance for screening,200 bp were cloned from the up and down sequences of HXT1 ORF respectively,then ligated to the 5′ and 3′ end of G418 resis-tance gene for recombination. After electroporation of GS115 spheroplast and screened through different G418 concentration plates,finally we obtained one HXT1 gene deletion mutant named GS115?HXT1. The growth rate and glucose consumption of this mutant were both lower than the wide type.

Effect of Specific Growth Rate and Nitrogen Source on the Production of Recombinant Lateolabrax japonicus Growth Hormone by Pichia pastoris / 微生物学通报

The bioreactor production of recombinant Lateolabrax japonicus growth hormone (rljGH) expressed intracellularly by Pichia pastoris was investigated. A strategy of feeding methanol at the exponential rate was established and the effect of specific growth rate on the rljGH production was examined. The results indicated that the average specific production rate increased and the rljGH production duration decreased as the specific growth rate increased. The maximum specific rljGH production (0.58 mg/g WCW) was achieved at a specific growth rate of 0.029/h. The effect of supplementing ammonium sulfate, peptone and yeast ex- tract on the rljGH production was further investigated. The results indicated that the effects of ammonium sulfate and peptone were not significant. Supplementing yeast extract of 2.5 g/L was advantageous for the rljGH production. The duration of the rljGH production was increased to 23 h from 17 h and the fermenta- tion stability of run-to-run could be improved.

Expression and Purification of Receptor Tyrosine Kinase ErbB2 Kinase Domain / 微生物学通报

The kinase domain of receptor tyrosine kinase(RTK) ErbB2 was expressed fused with GFP in Pichia pastoris. Recombinant expression vector pPIC3.5K was constructed in Escherichia coli TOP10. The right P. pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of recombinant vector, and then induced by methanol in baffled shake bottles. The strain with highest protein yield was scaled up in a 5 L fermentor. Recombinant protein was analyzed with tyrosine kinase assay after Ni2+ affinity chromatograph. Results showed that the 100 kD recombinant protein with tyrosine kinase activity was successfully expressed in P. pastoris.

Refolding of the fusion protein of recombinant enterokinase light chain rEKL / 生物工程学报

The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5% Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTP, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKL molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95%, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.

Detection of intracellular reactive oxygen species by flow cytometry in Pichia pastoris fermentation / 生物工程学报

In Pichia pastoris fermentation, methanol was oxidized into carbon oxide and produced a byproduct H2 O2, one of the partially reduced forms of molecular oxygen known as reactive oxygen species (ROS) . ROS are highly damaging towards cellular constituents. Flow cytometry (FCM) is an excellent method that permits the rapid, optical analysis of individual cells and has many advantages over conventional cytometry. However, its use in detecting intracellular ROS levels during Pichia fermentation was rarely reported. In our work, by means of flow cytometry, two fluorescent dye 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and propidium iodide (PI) were used to detect ROS. The effect of intracellular ROS on Pichia pastoris cells during fermentation was studied through the comparison between DCFH-DA/PI double-stained cells and PI single-stained cells. In this study, the loss of cell viability during fermentation was correlated with the accumulation of ROS. At the glycerol batch and fed-batch phase, little ROS was accumulated intracellularly and cell viability reached almost 100%. At the early methanol fed-batch phase, intracellular ROS accumulation took place but 98.5% cells still kept viable. At the later methanol fed-batch phase, 94.0% cells accumulated high ROS. As a result, some cells lost their viability because of the damage of ROS. 25.4% dead cells accumulated high ROS in the total 29.1% dead cells.

Localization of Hepatitis B Surface Antigen Within Recombinant CHO Cells in Response to Dimethyl Sulfoxide / 中国生物工程杂志

The intracellular hepatitis B surface antigen (HBsAg) content per cell was increased by 7.2-fold in the culture with 1.5% dimethyl sulfoxide (DMSO) compared with that in the control without DMSO, while the extracellular HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. Electron microscope has been employed to reveal large dilated structures within recombinant CHO cells in the presence DMSO. The dilated structures have a distribution within whole cytoplasm, and some dilated areas were engulfed in the nucleus. These large, dilated structures were not observed in the control. Immunogold labeling was used to discover the accumulated HBsAg was localized within these dilated areas, and some HBsAg-specific labels were detected in the nucleus membrane, owing to the encroachment of the dilated areas upon nucleus. The result could help to reveal the mechanism of intracellular HBsAg accumulation in the presence of DMSO.

Metabolism of recombinant CHO-GS cell reducing of toxic effect of ammonia / 生物工程学报

The toxic effect of ammonia on rCHO-GS cell decreased obviously due to the transfection of GS system in serum-free culture. The maximum cell density, 15.6 x 10(5) cells/mL was obtained in the culture with 1.42 mmol/L ammonia. The growth of rCHO-GS cell was inhibited with an increased ammonia concentration. However, a cell density of 8.9 x 10(5) cells/mL was obtained when the concentration of ammonia was 12.65mmol/L. The intracellar metabolic pathways were affected due to the decrease of the toxic effect of ammonia on rCHO-GS cell. With the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L, the yield coefficients of cell to glucose and lactate to glucose decreased. The activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) increased by 43%, 140% and 25%, respectively, indicating that the utilization of glucose increased and the glycolysis pathway was more prone to efficient energy metabolism pathway. An increased activity of glutamate-pyruvate aminotransferase (GPT) showed that the conversation from glutamate to alpha-ketoglutarate was shifted to glutamate-pyruvate transamination pathway. The deamination pathway was inhibited due to a decreased activity of glutamate dehydrogenase. In addition, the number of cells in G0/G1 phase increased and the specific production rate of recombinant protein increased by 2.1-fold with the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L.

Microbial transformation of sinenxan A, a rich constituent in callus cultures of Taxus / 药学学报

<p><b>AIM</b>To study the microbial transformation of sinenxan A.</p><p><b>METHODS</b>Choose two strains of Fungi (Mucor spinosus AS 3.3450 and Cunninghamella echinulata AS 3.3400) and a strain of bacterium (Proteus vulgaris AS 1.1208) to transform the substrate.</p><p><b>RESULTS</b>Three products were obtained and identified as 10-deacetylsinenxan A1, 6 alpha-hydroxy-10-deacetylsinenxan A2 and 9 alpha-hydroxy-10-deacetylsinenxan A3 respectively.</p><p><b>CONCLUSION</b>Sinenxan A is facile to be transformed by microorganisms, the 10-acetyl group of which is an active group.</p>

Fermentation behaviors of recombinant Pichia pastoris under inhibited methanol concentration / 生物工程学报

Chemostat culture was performed to characterize the growth, substrate consumption and the hirudin production, and to disclose their interrelations in the fermentation of recombinant Pichia pastoris. The Andrew substrate-inhibited growth model is more suitable than Monod model to simulate the growth of Pichia pastoris on methanol. Therefore, two stationary states can be obtained in the continuous culture at a certain dilution rate because of the substrate inhibition on cell growth. The stationary state could be obtained if only the dilution rate not more than 0.048 h(-1) in the continuous fermentation. The concentrations of cell, methanol and hirudin were constant after 50 h continuous culture with dilution rate at 0.04 h(-1). However, it could not be obtained when the dilution rate more than 0.048 h(-1) because the other stationary point at S > 0.048 h(-1) is unstable. Therefore, it was found that the cell concentration declined and the methanol concentration increased from 2.9 g/L to 18.1 g/L within 18h at dilution rate 0.06 h(-1). Thus, the fed-batch culture with a constant specific growth rate was carried out to disclose the fermentation behavior at high and constant methanol concentration in aid of a methanol sensor. The theoretical maximum specific growth rate, microm = 0.0464 h(-1), was found under critical methanol concentration, Scrit = 3.1 g/L. The growth of P. pastoris was typically methanol-limited at the methanol concentration S < Scrit. It was, however, inhibited at S > Scrit. The maximum specific Hir65 production rate qp was obtained at 0.2 mg/(g x h) when methanol concentration and mu were 0.5 g/L and 0.02 h(-1), respectively. The specific Hir65 production rate qp increased with the increase of mu and S at mu < 0.02 h(-1), and decreased at mu > 0.02 h(-1). The specific methanol consumption rate increased with the increase of S when S < 5 g/L, but decreased when S > 5 g/L. At last, the high Hir65 production rate 0.2 mg/(g x h) was obtained in the fermentation conducted under methanol-limited concentration and mu controlled at 0.5 g/L and 0.02 h(-1), respectively, while the specific methanol consumption rate is low only at 0.04 g/(g x h), showing the potential for the strategy of getting high Hir65 production rate at the low consumption of methanol.

Effects of different methanol feeding strategy on hirudin production in high-density fermentation by recombinant Pichia pastoris / 生物工程学报

Four different methanol feeding modes were evaluated in the hirudin production in high-density fermentation by Pichia pastoris. It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level. On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography. However, the stable methanol concentration was perfectly achieved by the on-line monitoring with methanol sensor. The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase. Therefore, the high cell dry weight (162 g/L) and high hirudin activity (2.4 x 10(4) ATU/mL or 1.7 g/L) was obtained in the fed-batch fermentation of recombinant Pichia pastoris by methanol-glycerol mixed feeding.

Application of Flow Cytometry in Viability Detection of Pichia pastoris Cells / 微生物学通报

Cell viability of Pichia pastoris was detected by flow cytometry (FCM) with two reagents fluorescein diacetate (FDA) and propidium iodide (PI). Compared with FDA/PI double-stained dot plots and PI single-stained dot plots,the latter could divide dead and living cells into two separate zones,and get the correct proportion. Then PI single-stained method was used to detect the change of cell viability in Pichia patoris fermentation. At glycerol batch and fed-batch phase,little dead cells were detected. At methanol fed-batch phase,cell viability decreased when cell weight increased,and was only 73.8% at 88 h.

Growth,Siderophore Production and Outer Membrane Protein Expression of Vibrio alginolyticus by Iron Regulation / 微生物学通报

Iron uptake mechanism of Vibrio alginolyticus was primarily investigated. V.alginolyticus could survive in the medium with high-concentration iron chelator. The strain of V. alginolyticus isolated from diseased fish produced more siderophore than that from marine environment. The extract of siderophore from V. alginolyticus could stimulate the growth of Escherichia coli mutant AN93. Under iron limitation,the growth rate was decreased and several outer membrane proteins were induced. Adding iron into the iron-limited medium the normal growth could be recovered.

OPTIMIZATION OF THE MEDIUM AND PROCESS PARAMETERS IN BATCH AMINOGLYCOSIDE ANTIBIOTIC JI-20A FERMENTATION / 微生物学通报

The medium and process parameters were optimized in batchaminoglycoside antibiotic JI-20A fennentation. The optimal medium consists mainly of comstarch 60g/L, peanut meal 30g/L, com slurry 8g/L, maltose 10g/L, inorganic compound and amylase moderate , methionin 1g/L and cobalt chloride 6?g/mL. It was significant to adjust medium pH after autoclaving and oxygenate the fennentation medium for the cell growth and JI-20A product.

Application of Direct Gene Disruption Method in Recombinant Pichia pastoris / 微生物学通报

Proteolytic degradation has been a severe problem when Pichia pastoris is employed to express recombinant proteins.One alternative method to circumvent this problem is to construct protease gene disruptant.However,the main study of gene disruption is focused on nonrecombinant Pichia pastoris rather than recombinant strain.In our study,we established two different methods to directly disrupt PRC1 and KEX1 gene in recombinant Pichia pastoris.On the basis of this,we further discussed and compared the application and advantages of both methods.