rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

Abstract

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast alpha-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN-, Br- and Cl-. On the basis of the estimated K(m) and kcat values it is evident that the pseudohalide SCN- is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

Figure 1. Expression of secreted rhEPO

(A) SDS/(12%) PAGE of supernatant from cultured P. pastoris expressing rhEPO, induced in BMMY (buffered-methanol complex medium) for 0, 24, 48 and 72 h. (B) Western blot analysis of secreted proteins at 72 h after methanol induction. MW, molecular mass.

Figure 2. Purification of rhEPO

(A) Gel filtration cromatography on a Sephadex G-75 column equilibrated and eluted with 0.1 M phosphate buffer (pH 6), 10 mM β-mercaptoethanol and 10 μM PMSF at a flow rate of 40 ml/min. The separation was monitored by measuring the absorbance at 280 nm. The fractions corresponding to the major peaks were collected and pooled for further analysis as indicated on the chromatogram. The inset shows SDS/PAGE of the rhEPO-containing G-75 pool 1. (B) Gel filtration chromatography of the rhEPO-containing G-75 pool 1 on a SMART system. A Superdex 75 PC 3.2/30 column was equilibrated and eluted with 10 mM phosphate buffer (pH 6.5) at a flow rate of 40 μl/min. The separation was monitored by recording the absorbance at 280 nm and a LMW calibration kit was used to estimate the molecular mass of the oligomeric enzyme. The inset shows glycoprotein detection in the rhEPO-containing G-75 pool 1. (C) SDS/PAGE and Coomassie Blue staining of purified rhEPO after Q-Sepharose anion-exchange chromatography. Ctrl, control; HRP, horseradish peroxidase; MW, molecular mass; stds, standards.

Figure 3. Absorption spectrum of purified rhEPO

Measurements were carried out at 25 °C in 0.1 M phosphate buffer, pH 7.

Figure 4. Single-wavelength kinetic traces of rhEPO-dependent oxidation of TNB in the presence of SCN⁻, Br⁻ and Cl⁻

Purified rhEPO (200 μl) was diluted to a final volume of 1 ml in a reaction mixture containing 0.1 M phosphate buffer (pH 7), 50 μM TNB and 150 μM NaSCN, NaBr or NaCl. Reactions were initiated by the addition of 30 μM H₂O₂ and DTNB formation was monitored spectrophotometrically at 412 nm as described in the Materials and methods section. Values were corrected for DTNB production in the absence of pseudohalides [37]. In the presence of Cl⁻, Br⁻ and SCN⁻ the rate of DTNB formation (−ΔA₄₁₂/min) was 0.0008, 0.004 and 0.016 (±S.D. 0.001) respectively.

Figure 5. Effect of substrate concentration on the activity of rhEPO

Lineweaver–Burk plot of DTNB formation versus substrate concentration. Reactions were started by the addition of 30 μM H₂O₂ in 0.1 M phosphate buffer (pH 7) containing 20 nM purified rhEPO, 50 μM TNB and the indicated concentrations of SCN⁻ and Br⁻. The change in absorbance measured at 412 nm was used to calculate DTNB production using ϵ₄₁₂=27.000 M⁻¹·cm⁻¹ [37]. The rate of oxidant production was determined over the first 20 s and corrected for TNB oxidation in the absence of anionic substrates. E₀, total enzyme concentration; v, rate of DTNB formation (μM/s).