Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Abstract

Background Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM). Results ImageJ analysis of the HIP’s SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones. Conclusions CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…For example, mutants of the α-MF—where at position 42, amino acid residue Leu is replaced by Ser, and at position 83, Asp is substituted with Glu—increase the productivity of the recombinant protein by almost threefold as compared with the wild type [ 52 ]. Deletions in the α-MF pro-region (Δ57–70) have improved the efficiency of expression of some heterologous genes in K. phaffii cells by almost an order of magnitude [ 100 , 101 , 102 ].…”