Pichia pastorisas a host for secretion of toxic saporin chimeras

Abstract

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino-terminal fragment of human urokinase (ATF) fused to the plant ribosome-inactivating protein saporin could be recovered. ATF-saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti-saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport-competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon-usage optimization greatly increased the expression levels of active saporin but not those of an active-site mutant SAP-KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine-tagged ATF-saporin chimera showing an IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras.

Reductive Activation of Type 2 Ribosome-inactivating Proteins Is Promoted by Transmembrane Thioredoxin-related Protein

“…Saporin is a plant toxin belonging to the type 1 family of RIPs that lack a B chain and bind cell surfaces with difficulty, although in the case of saporin it was described that it can bind the ␣2 macroglobulin receptor unequally distributed in different types of cells (12). Intracellular trafficking of saporin has not been fully elucidated yet, but it does not require intracellular reduction to intoxicate the target cell.…”

Biotechnological Production of Recombinant Tissue Plasminogen Activator Protein , its Application

“…The instability of the protein may be due to the effect of proteases, which are also secreted from Nevertheless, the rt-PA yield was significantly higher than the tPA production (up to 0.017%) in a hairy root system (54). Unlike tPA, reteplase has no carbohydrate side chains, and thus can be produced in E. coli cells, but most of the protein is present as an inclusion body, making its extraction and renaturation a time to consume downstream process, involving solubilization and protein refolding and dialysis (55). The culture medium could be optimized e.g.…”

Engineering Aggregation Resistance in IgG by Two Independent Mechanisms: Lessons from Comparison of Pichia pastoris and Mammalian Cell Expression

“…While the inclusion of the spacer generally enhances the cleavage efficiency of this endopeptidase specific for dibasic sites, several studies have demonstrated problems with the subsequent proteolytic processing of the EA repeats. [54][55][56]76 Apparently, the quantity and/or activity of the STE13-encoded DPAPase A in the secretory pathway is not sufficient to process these repeats for large amounts of recombinant protein expressed under strong promoters. The resulting secretion of molecules with amino-terminal EAEA extensions was also observed for both the heavy and the light chains of the initial IgG constructs expressed in the Pichia system in this study.…”