Pichia pastoris as a host for secretion of toxic saporin chimeras.
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.
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, via Bassini 15, Milan, Italy.
Alessio
A
Lombardi
Sara
S
Bursomanno
Teresa
T
Lopardo
Roberta
R
Traini
Marco
M
Colombatti
Rodolfo
R
Ippoliti
David J
DJ
Flavell
Sopsamorn U
SU
Flavell
Aldo
A
Ceriotti
Maria Serena
MS
Fabbrini
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D000922:Immunotoxins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics* / Q000633:toxicity; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000633:toxicity; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000633:toxicity; D054789:Ribosome Inactivating Proteins, Type 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000633:toxicity; D000076985:Saporins; D014170:Transformation, Genetic; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics* / Q000633:toxicity
D003062:Codon; D017931:DNA Primers; D000922:Immunotoxins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C109077:urokinase-ATF-SAP-3, recombinant; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D014568:Urokinase-Type Plasminogen Activator