Heterologous expression of hagfish thread proteins EsTKα and EsTKγ in Pichia pastoris and in vitro self-assembly into an ordered fibrous network

<i>Eptatretus stoutii</i> thread keratin α and γ (EsTKα and EsTKγ) are essential structural components conferring hagfish slime with exceptional strength and toughness. These proteins exhibit exceptional mechanical properties, rendering them promising candidates for biomimetic material applications. However, the ability of conventional prokaryotic expression systems to produce these proteins is notably limited, as they often fail to achieve proper folding and post-translational modifications. In this study, <i>Pichia pastoris</i> strains with various phenotypic characteristics were screened, and key parameters, including signal peptides and fermentation conditions, were systematically optimized. The SMD1168H strain was identified as an optimal host for recombinant protein production. Following methanol induction in a 5-L fermentor, extracellular expression yields reached 350.0 mg/L for EsTKα and 385.0 mg/L for EsTKγ. The conformational changes in EsTKα and EsTKγ during in vitro self-assembly were characterized using multiple analytical techniques, which confirmed successful self-assembly into an ordered fibrous network. This study reports the successful heterologous expression of EsTKα and EsTKγ in the eukaryotic host <i>P. pastoris</i>, highlighting the potential of <i>P. pastoris</i> as a robust platform for the scalable production of these structurally complex proteins.