The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine 
kinase receptor overexpressed in 30% of human breast cancers. One of the 
mechanisms by which tumor cell proliferation can be inhibited consists in 
hampering HER2 dimerization by targeting its extracellular domain with specific 
antibodies. In recent clinical practice, a valuable alternative to entire IgGs 
resides in the use of smaller molecules, such as single-chain variable fragments 
(scFv), developed for selective molecular targeting. In this paper, we report on 
the production and purification of a soluble anti-HER2 scFv antibody secreted by 
Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at 
the 3'-end was inserted into the expression vector pPICZα and transformed in P. 
pastoris. The highest expression level was obtained in presence of 0.5% methanol 
and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. 
pastoris proved very valuable as an expression system, allowing the isolation of 
10 mg/L of highly purified antibody, remarkably higher than previously reported 
data. The functionality of purified anti-HER2 scFv was assessed by 
cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer 
cells, showing good affinity and high selectivity for the target membrane 
receptor. These findings confirm that P. pastoris is a suitable host for high 
level expression of antibody fragments and highlight the potential role of 
scFv800E6 in diagnostic and therapeutic application.