Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA)expressed in Pichia pastoris - Correct N-terminal processing and secretionof heterologous proteins expressed using the PHA-E signal peptide
Rjm. Raemaekers et al., Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA)expressed in Pichia pastoris - Correct N-terminal processing and secretionof heterologous proteins expressed using the PHA-E signal peptide, EUR J BIOCH, 265(1), 1999, pp. 394-403
Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and
snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pich
ia 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 r
ecombinant lectins, purified by affinity chromatography, agglutinated rabbi
t erythrocytes at concentrations similar to the respective native lectins.
However, incomplete processing of the signal sequence resulted in PHA-E, PH
A-L and GNA with heterogenous N-termini, with the majority of the protein c
ontaining 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 t
he alpha-factor preprosequence led to efficient processing N-terminal to th
e Glu-Ala sequence, but inefficient removal of the repeats themselves, resu
lting in polypeptides with heterogenous N-termini still containing N-termin
al extensions. In contrast, PHA expressed with the native signal peptide wa
s secreted, correctly processed, and also fully functional. No expression o
f GNA from a construct containing the native GNA signal peptide was observe
d. The PHA-E signal peptide directed correct processing and secretion of bo
th GNA and green fluorescent protein (GFP) when used in expression construc
ts, and is suggested to have general utility for synthesis of correctly pro
cessed proteins in Pichia.