Strains, vectors and other reagents
The P. pastoris GS115, pPICZαA vector, and Zeocin antibiotic were obtained from Invitrogen (CA, USA). P. pastoris were grown in YPD medium containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L D-glucose. To prepare YPD plates, 2% agar (w/v) was added to the YPD medium. YPD-Zeocin plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar, and 0.1-0.2 mg/mL Zeocin) were used for selecting multicopy transformants. The P. pastoris cells were cultured in BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 400 μg/L biotin, and 0.1 M potassium phosphate, pH 6.0) for growth and in BMMY medium (1% yeast extract, 2% peptone, 400 μg/L biotin, 1% methanol, and 0.1 M potassium phosphate, pH 6.0) for induction. All primers were synthesized by Sangon Biotechnology Corp. (Shanghai, China). All restriction enzymes, DNA marker, synthesized genes (human BSA-UTI fusions) and protein marker were purchased from Takara (Dalian, China). The standard human UTI, trypsin purchased from Sigma-Aldrich (St. Louis, USA).
Construction of expression vector pPICZα-HSA-UTI
Construction of rh-bikunin expression vector was based on pPICZαA vector. The synthesized human serum albumin (HSA) gene containing different domains were added 6 × His tag, several Gly linkers and the recognition site of Enterokinase (EK) at the C-terminal (as shown in Figure ). The synthesized HSA gene and UTI gene were ligated to pMD18-T vector. The recombinant plasmids pMD-HSA-UTI were digested with Xho I and Not I and then inserted into the same site of pPICZαA. The recombinant expression plasmids were designated as pPICZα-UTI, pPICZα-D1-UTI, pPICZα-D2-UTI, pPICZα-D12-UTI and pPICZα-D123-UTI, respectively.
Figure 1 Maps of fusion genes. Plasmid pPICZαA was used as a parent vector for constructing these fusions. D1, D2 and D3 represent different domain of human serum albumin (HSA) gene. Several Gly residues designed as linker. EK represent the site of enterokinase. (more ...)
Transformation of P. pastoris and selection of high-level expression
The five Sac I restriction endonuclease linearized recombinant expression vectors were introduced into P. pastoris GS115 by electroporation using a Micropulser (Bio-Rad, USA) according to the manual, respectively. Transformed cells were selected by growth on yeast extract peptone dextrose (YPD) agar plates containing Zeocin (0.5 mg/mL). After the multicopy transformants appeared, single clone was cultured in 5 mL BMGY medium at 28°C with shaking at 250 rpm for 24 h. The cells were then centrifuged and resuspended in 5 mL BMMY medium to induce expression for 4 days. The culture medium (0.5 mL) was sampled per day and centrifuged at 4°C, 10,000 rpm for 5 min. Cell pellet and supernatant were separated. The supernatant was tested for UTI activity and cell pellet was used for genomic DNA analysis. pPICZα A blank plasmids were also transformed as a negative control.
Optimization of fermention conditions by shake-flask mode
In order to determine the effect of pH on the expression level of rh-UTI, the BMGY media was performed at different pH values (pH 4.0, 5.0, 5.5, 6.0, 6.5, 7.0). In order to determine the effect of feeding mode on the expression level of rh-UTI, inorganic salt, peptone, yeast extracts and glycerol was used as the carbon sources in BMGY media by continuous fed-batch mode, respectively.
A stock culture of P. pastoris was grown to an A600 of 3-6 in a 5-L shake flask containing 2 L YPD. The shake flask culture was used to inoculate an 30-L fermenter (Bioengineering, AG) containing 20 L of fermentation basal salts medium FM22 supplemented with PTM1 trace salts (1.1 mL of stock solution/L) and biotin (0.4 mL of the stock solution/L). The dissolved oxygen level (DO) was set at 30% and the stirring rate was 700 rpm. The pH of the medium was maintained 6.0-6.5 by automatic addition of 5 N NH4OH and 1 M phosphoric acid and 5% antifoam as required. Temperature was maintained at 30°C. The initial cultivation terminated when all glycerol was consumed (about 14 h) at batch phase. Continuous 2% peptone and 1% yeast extract feeding were carried out for about 6 h in the subsequent fed-batch phase. To induce rh-UBI expression, 100% methanol was fed at 3 mL/h/L for 50 h. Sampling of the culture medium at the end of each phase was performed for assay of rh-bikunin activity.
Purification of rh-UBI and assay of trypsin inhibitory activity
The supernatant was collected by centrifugation, 5 M NaCl and phosphate buffer was added to a final concentration of 1 M and 50 mM, respectively. Finally, the pH value was also adjiusted to 7.4 with NaOH. Then the treated supernatant was was clarified with a 0.45 μm cellulose membrane. The supernatant was purified with chelating sepharose affinity chromatography and Q sepharose F. F. anion exchange chromatography in turn. Then after digested by enterokinase, the sample was isolated and purified by chelating sepharose affinity chromatography and SP sepharose F.F cation exchange chromatography. The purity of each step protein sample was checked by SDS-PAGE. The final rh-bikunin protein was stored at -20°C for further assay of trypsin inhibitory activity accroding to previous study ([Page, Quillien et al. 2000
]; [Wang, Yan et al. 2008
Carbohydrate digestion and amino-terminal sequence
The amino-terminal sequence of rh-bikunin was determined automated Edman degradation method (Shanghai Sangon Biological Engineering Technology & Services CO., Ltd). N-linked glycosylation was assayeded by digestion of the protein with N-glycosidase F. Protein samples (20 μg) were boiled for 5 min in 1% (w/v) SDS and 50 mM DTT. Samples were then diluted to 0.1% SDS, 5 mM DTT, 20 mM sodium phosphate (pH 7.4), 25 mM EDTA, and 2% Triton X-100. N-glycosidase F (0.8 U) was added and samples were incubated at 37°C overnight. Deglycosylation of the protein was assessed by a shift in electrophoretic mobility on SDS-PAGE analysis.