Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.
Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.
Raemaekers
R J
RJ
Department of Biological Sciences, University of Durham, UK.
de Muro
L
L
Gatehouse
J A
JA
Fordham-Skelton
A P
AP
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D027801:Galanthus; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006031:Glycosylation; D037102:Lectins / Q000096:biosynthesis* / Q000235:genetics; D037241:Mannose-Binding Lectins; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010835:Phytohemagglutinins / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D037121:Plant Lectins; D010940:Plant Proteins; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020539:Sequence Analysis, Protein
D006023:Glycoproteins; D037102:Lectins; D037241:Mannose-Binding Lectins; D010455:Peptides; D010835:Phytohemagglutinins; D037121:Plant Lectins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C067494:erythroagglutinating phytohemagglutinin; C101135:phytohemagglutinin L protein, Phaseolus vulgaris; C063117:snowdrop lectin; D000072235:Mating Factor