Construction and screening of balanced Agkisacutacin expression strains

The coding sequence of the α- or β-subunit of Agkisacutacin was inserted into the Xho I- Not I sites of the pPIC9 or pUCZR vectors and the resulting constructs were designated pPIC9/α or pUCZR/β, respectively (Fig. 1). The pPIC9/α and pUCZR/β vectors contain the functional histidinol dehydrogenase (HIS4) and rDNA repeat genes, respectively, for homologous recombination into the genome of P. pastoris. Both the α- and β-subunit coding genes were fused with the Saccharomyces cerevisiae α-mating factor signal sequence and placed under the regulation of an AOX1 promoter. To generate the balanced expression strain, the Agkisacutacin α-subunit expressing plasmid was transformed into P. pastoris strain GS115 and selected using histidine-deficient minimal dextrose (MD) plates. The colony with the highest α-subunit expression was selected by SDS-PAGE (Fig. 2a) and subjected to further transformation with the β-subunit-expression vector pUCZR/β. The α- and β-subunit co-expression colonies GS115/αβ were screened using histidine-deficient MD plates containing 600 μg/ml Zeocin and the expression of αβ heterodimeric proteins was monitored by Western blot using anti-Agkisacutacin monoclonal antibody 1B9 (Fig. 2b). The colony with the highest relative expression was selected for further balanced expression screening by stepwise increases in the drug concentration with 600 μg/ml, 800 μg/ml and 1000 μg/ml Zeocin on histidine-deficient MD plates. The expression of each subunit individually and the αβ heterodimer in the resulting colonies was determined by SDS-PAGE under reducing (Fig. 2c) and non-reducing (Fig. 2d) conditions, respectively. The expression of the α- and β-subunits gradually shifted from more α-subunit than β-subunit to more β-subunit than α-subunit when the concentration of Zeocin was increased from 600 μg/ml to 1000 μg/ml (Fig. 2c). With 800 μg/ml Zeocin, the expression of the α- and β-subunits reached a balance, with nearly equal amounts (Fig. 2c). The highest expression of αβ heterodimer (Fig. 2d) was also found in colonies with balanced expression of the α- and β-subunits. The strategy of combining the mutant histidinol dehydrogenase gene (his4) and rDNA repeat loci for homologous recombination and stepwise drug screening achieved a successful result for balancing the expression of rAgkisacutacin in P. pastoris.

Figure 1 Strategy for the construction of rAgkisacutacin expression strains. This schematic map represents the constructed expression vectors for the Agkisacutacin α- and β-subunits, designated pPIC9/α and pUCZR/β, respectively, in which, his4 and rDNA non-coding sequences allow the vector to be inserted into the corresponding sites in the genome of strain GS115 through homologous recombination. The pUCZR vector was constructed by introducing a non-coding rDNA (indicated as rDNAnc) sequence into the pPICZα vector. The expression of both α- and β-subunits was under the control of the AOX1 promoter. Full size image

Figure 2 Establishing balanced expression strains for rAgkisacutacin subunits. (a) The expression of the α-subunit in transformants, designated GS115/α, was examined by 15% SDS-PAGE under reducing conditions and colony with the highest expression was selected for further transformation with the β-subunit expressing vector. (b) The expression of the αβ heterodimer from the transformants, designated GS115/αβ, was examined by Western blot and the colony with the highest expression was used for further drug screening. Ctr.: colony transformed with empty vectors. The expression of the α- and β-subunits from screened colonies with various doses of Zeocin was examined by SDS-PAGE under reducing conditions (c) or by Western blot under non-reducing conditions (d). No.: colony number. Full size image

Expression condition optimization

The optimal culture conditions, including pH and temperature, for protein expression were determined using shake flasks. The best condition for rAgkisacutacin expression was at pH 5.0 (Fig. 3a). The optimal temperature range was from 25 °C ~ 30 °C (Fig. 3b). Neither pH nor temperature had a significant effect on the biomass in the cultures (Fig. 3a,b).

Figure 3 Optimization of culture conditions for rAgkisacutacin expression in flasks. Protein expression (upper panel) and the corresponding biomass (lower panel) in shake flasks at indicated pH (a) or temperatures (b) were examined. Three individual colonies were tested for each condition. Full size image

Pilot-scale fermentation of rAgkisacutacin

The typical fermentation process (Fig. 4a) was composed of three steps: a batch phase, glycerol feeding phase and methanol induction phase. During the batch phase, the yeast seeds were cultured with BMGY medium containing 4% glycerol (pH 5.0) and controlled at 28 °C. The batch phase usually lasted 17–18 h and ended when the wet cell weight (WCW) reached 140 g/L and a sharp dissolved oxygen (DO) spike occurred, indicating the depletion of glycerol in the culture medium. During the glycerol feeding phase, 50% glycerol supplemented with 12 ml/L PTM1 solution was supplied through feeding and DO was controlled at 30% by limiting airflow. The temperature was maintained at 28 °C and pH remained at 5.0. This process usually lasted 7–8 h and ended when the WCW reached approximately 250 g/L. After feeding stopped and a DO spike was observed, the methanol induction phase was started by a stepwise increase in the methanol feeding rate (100% methanol with 12 ml/L PTM1 salts) from 21.6 ml/min to 64.8 ml/min over 4–5 h. The DO was restricted to approximately 30% by limiting the supply of methanol and oxygen (Fig. 4a). The methanol induction phase lasted 45 h and ended when the yeast WCW reached approximately 400 g/L (Fig. 4b). The accumulated rAgkisacutacin increased progressively during the first 35 h of methanol induction; however, prolonged cultivation led to either stagnation or a decline in both cell growth (Fig. 4b) and rAgkisacutacin production (Fig. 4c). Moreover, the levels of the α- and β-subunits remained equal throughout the fermentation process (Fig. 4d).

Figure 4 Pilot-scale fermentation of rAgkisacutacin. (a) The plotted parameters show the DO, pH, temperature and the feeding speed for glycerol and methanol during a typical three-step fermentation process with a 14-L New Brunswick BioFlo 115 fermentor. (b) The growth rate was measured and presented as WCW during the fermentation process. rAgkisacutacin expression during the fermentation was examined by Western blot with anti-Agkisacutacin antibody 1B9 (c) and by SDS-PAGE stained with Coomassie blue (d) under reducing (lower panel) and non-reducing (upper panel) conditions. Full size image

Purification and characterization of rAgkisacutacin

The downstream processing of rAgkisacutacin fermentation was composed of three steps (Fig. 5a): clarification of the broth by centrifugation and filtration, capturing rAgkisacutacin by ion-exchange chromatography and formulation by molecular-exclusion chromatography. The products from each step were monitored by SDS-PAGE (Fig. 5b). Using this process, the final products surpassed 95% purity as determined by HPLC (Fig. 5c). Additionally, both the α- and β-subunits of rAgkisacutacin were characterized by LC-MS, which showed the sequence coverage of most of the proteins and which confirmed that the sequence of rAgkisacutacin exactly matched that of nAgkisacutacin (Fig. 5d). Thus, an effective downstream purification process was established for rAgkisacutacin production.

Figure 5 Downstream processing and characterization of rAgkisacutacin. (a) Schematic presentation of the workflow for downstream processing. The culture supernatant was collected and clarified by centrifugation and stepwise filtration. After diluting the sample to adjust the pH and salt concentration, rAgkisacutacin protein was captured by CM FF and further purified using S-100 HR chromatography. (b) The samples from each step during the purification processes were monitored by 15% SDS-PAGE. S: the supernatant from centrifugation; 0.45/500/10 FT: the flow-through of filtration through 0.45 μm/500 kDa/10 kDa hollow-fibre membranes; CM: CM FF column; S-100: S-100 HR column; M: pre-stained protein molecular marker. (c) The purity of rAgkisacutacin was analysed by SCE. (d) Approximately 20 μg of final purified rAgkisacutacin was analysed by 15% SDS-PAGE under reducing and non-reducing conditions as indicated and was visualized by Coomassie blue staining. The bands corresponding to the α- and β-subunits were excised and digested for LC-MS analysis. The amino acid sequences of the α- and β-subunits are listed and the detected peptides from LC-MS are underlined and bolded. Full size image

Biological activity of rAgkisacutacin

The binding affinity of rAgkisacutacin to solid-phase rGPIb was determined and compared with nAgkisacutacin by ELISA (Fig. 6a). Both rAgkisacutacin and nAgkisacutacin bound to rGPIb in a concentration-dependent manner, with extremely similar half-maximal binding concentrations of 6.5 ± 0.1 ng/ml and 6.1 ± 0.5 ng/ml, respectively (Fig. 6a). The ex vivo FACS assay with human whole blood showed that the binding activities of rAgkisacutacin and nAgkisacutacin were 79.4% and 86.5%, respectively (Fig. 6b). Antiplatelet activity was measured by ristocetin-induced platelet aggregation assay. Both rAgkisacutacin and nAgkisacutacin showed dose-dependent antiplatelet activity and nearly complete reduction of aggregation with rates of 5.65% and 3.46%, respectively at the 3 μg/ml dose (Fig. 6c). These data consistently indicate that rAgkisacutacin possesses extremely similar antiplatelet activity to that of nAgkisacutacin.