[Molecular chaperones influence the expression of single-domain antibodies in Pichia pastoris]

Single-domain antibodies (sdAbs) have garnered increasing attention in the biomedical field due to their low molecular weights and exceptional tissue penetration capacity. Compared with mammalian cell and Escherichia coli expression systems, the Pichia pastoris expression system offers advantages such as low costs, short production cycles, and robust support for protein folding. To enhance the expression efficiency of sdAbs in yeast, we constructed a co-expression system in P. pastoris involving six molecular chaperones (HDPI, YPDI, BZIP, TO, RPPO, and Sec) and the target sdAbs. The effects of these chaperones on the structural and functional properties of sdAb were systematically evaluated through circular dichroism (CD) spectroscopy for secondary structure analysis, measurements of thermal melting temperature (T_m) and concentration of urea when 50% protein unfolded (CUU), turbidity assays, and trypsin digestion experiments. The results demonstrated that: Co-expression of Sec significantly increased the expression level of sdAbs by 1.68 times; None of the chaperones significantly altered the secondary structure or antigen-binding activity of the sdAbs; Only the BZIP co-expression group showed enhanced thermal stability, with a Tm increase of 2.4 ℃; The YPDI group exhibited markedly improved resistance to urea-induced denaturation, with a 1.2 mol/L increase in CUU; In trypsin digestion assays, the YPDI group displayed the highest stability, retaining 58.3% of the enzyme activity after 3 h; Turbidity assays indicated that YPDI and BZIP effectively suppressed antibody aggregation, whereas HPDI, TO, and RPPO promoted aggregation. In conclusion, this study reveals distinct regulatory effects of different molecular chaperones on the physicochemical properties of sdAbs, providing a theoretical foundation and practical insights for optimizing the production processes of sdAbs.