Expression, purification and characterization of recombinant human β-amyloid 1–42 in Pichia pastoris

Abstract

The human peptide rhAβ 1–42 was effectively produced through a novel expression system and purification procedure. The peptide rhAβ 1–42 was successfully expressed in Pichia pastoris, the methylotrophic yeast that has never been used as host. The cDNA encoding full-length hAβ 1–42 was synthesized with yeast bias codons and cloned into the pPICZαA vector in frame with the yeast α-factor secretion signal under the transcriptional control of the AOX1 promoter and integrated into the secreting expression organism P. pastoris strain X33. Production of rhAβ 1–42 through fermentation was further optimized and scaled up in an 80 L fermentor. Secreted rhAβ 1–42 was purified using a two-step purification scheme: SP Sepharose ion exchange chromatography and source™ 30 RPC. The purification procedure is fast and efficient and reached a recovery of >93% without loss of activity. The purified rhAβ 1–42 was confirmed by Western blotting analysis and N-terminals amino sequencing analysis. This efficient and cost-effective expression system facilitates large-scale production and purification for recombinant rhAβ 1–42.