Methanol-Free Protein Expression in Komagataella phaffii With Magnetic or Non-Magnetic Heating

Abstract

Komagataella phaffii is among the most widely used expression systems, with methanol-inducible promoters being preferred for protein expression due to their stringent regulation and exceptional strength. However, the applicability of this system, particularly in food and pharmaceutical products, is limited by methanol's toxic and pro-inflammatory properties. Therefore, obtaining a novel methanol-free expression system is necessary. In this study, we obtained a novel expression plasmid, pHSPαA, carrying the HSP70 promoter (P_HSP70) to regulate heterologous expression through heat induction. The extracellular expression of azurin was achieved using this methanol-free system under the control of P_HSP70, induced by either magnetic or non-magnetic heating. To enhance heat-induced expression, recombinant cells were immobilised with Fe₃O₄@PEI_{25 kDa} nanoparticles, which facilitated heat release under an AC magnetic field, thereby increasing cell permeability and protein secretion. A time-dependent increase in protein expression was observed in non-magnetic heating but not under magnetic heating. However, immobilised cells exhibited a higher protein secretion capacity compared to non-immobilised cells. These findings suggest that the novel methanol-free expression system represents a promising alternative for heterologous gene expression, particularly for the production of therapeutically relevant and food-grade recombinant proteins.

Keywords: Komagataella phaffii; HSP70 promoter; alternating magnetic field; magnetic nanoparticles; methanol‐free expression.

FIGURE 1

Schematic representation of the study. (1) Obtaining recombinant DNA and transformation of yeast cells. (2) Magnetic immobilisation of recombinant yeasts. (3) P _HSP70 induction by magnetic or non‐magnetic heating. (4) Analysis of recombinant proteins.

FIGURE 2

Spotting test result of recombinant Komagataella phaffii cells exposed to 39°C and 42°C (A). Protein expression by non‐magnetic heating (B). Western blot image of extracellular azurin expression (C) and azurin expression level (D). C: Positive control (azurin expression by methanol induction), M: Protein marker, 1, 2, 3, 4 and 5: 24, 48, 72, 96 and 120 h of azurin expression, respectively). ***p < 0.001.

FIGURE 3

Characterisation of MNPs. TEM images (A), VSM graph (B), temperature–time graph of MNPs under an AC magnetic field (C), and comparison of VSM and SAR values (D).

FIGURE 4

Magnetic immobilisation of Komagataella phaffii cells (A). Images of yeast culture and magnetically immobilised cells before and after exposure to a magnet (B). The immobilisation efficiency of recombinant cells (C). SEM and pseudo‐colour SEM images of non‐immobilised and magnetically immobilised cells (D). 72 h toxicity results of magnetically immobilised cells (spotting test images and OD₆₀₀ measurement graphs) (E). *p < 0.05; ***p < 0.001.

FIGURE 5

Results of 72 h toxicity of magnetic heating on yeast cells (spotting test images and OD₆₀₀ measurement graphs) (A). Protein expression by magnetic heating (B). Western blot image of extracellular azurin expression (C). The azurin expression level (D). C: Positive control (azurin expression by methanol induction), M: Protein marker. 1, 2, 3, 4 and 5; 24, 48, 72, 96 and 120 h, respectively. ***p < 0.001.

FIGURE 6

Extracellular recombinant azurin production by magnetic or non‐magnetic heating. Western blot image of azurin expression (A). The azurin expression level (B). 1 and 2, respectively, culture liquid from the cells immobilised with MNPs containing 50 and 125 ppm Fe and induced by magnetic heating, respectively; 3, pHSPαA‐Azu/X‐33 and 4, pHSPαA/X‐33 culture liquids from incubated at 30°C, M: Protein marker; 5, 6 and 7, culture liquid from the cells immobilised with MNPs containing 0 ppm (non‐immobilised), 50 ppm and 125 ppm Fe and induced by non‐magnetic heating, respectively. ***p < 0.001.