Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae

Abstract

Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the E N 0 1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Dde1, which has a ssll mutation causing a supersecretion of human lysozyme [Suzuki, [58][59][60][61][62][63][64], was 20-fold grcater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found thc proccssed C-tcrminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.

Site‐Directed Mutagenesis and Sugar‐Binding Properties of the Wheat Germ Agglutinin Mutants Tyr73Phe and Phe116Tyr

“…Authentic WGA was purchased as a mixture of three isolectins from Honen, and the individual isolectins were purified from this mixture by using a cation-exchange column (Mono S HR5/5, Pharmacia) as described previously (Nagahora et al, 1992). Rabbit anti-WGA serum and goat anti-(rabbit IgC)-alkaline-phosphatase conjugate were obtained from Sigma and Cappel, respectively.…”

“…All other reagents were biochemical reagent grade. The Escherichia coli and Saccharomyces cerevisiae strains used in the experiments were those described in a previous paper (Nagahora et al, 1992).…”

“…Mutant prepro-WGAZ genes were excised fr-om the M13mp19 vector as a SalI-Hind111 fragment and were inserted into SalVHindIII-digested plasmid pNJl053 containing the yeast EN01 promoter (Nagahora et al, 1992) to give mutant prepro-WGA2 expression plasmids containing the yeast LEU2 gene as an auxotrophic marker. To construct the expression plasmid containing the yeast URAS gene as an auxotrophic marker, the EcoRI -Hind111 fragments containing the E N 0 1 promoter and mutant or wild-type prepro-WGA gene were excised from the above expression plasmid or pSWC (Nagahora et al, 1992), and they were inserted into the EcoRYHindIIT-digested plasmid YEp352 (Hill et al, 1986) to give a mutant or wild-type prepro-WGAZ gene expression plasmid (pSWC-URA) containing the yeast URAS gene as an auxotrophic marker.…”

“…S. cerevisiae strain leu2,uru3, his1 o r his3, ssll) (Suzuki et al, 1989;Nagahora et al, 1992) was transformed either with a mutant or wild-type WGA2 gene expression plasmid harboring the LEU2 gene or with a plasmid harboring the URA3 gene, according to the method of Ito et al (1983). The leucine or uracil prototroph was selected and grown at 30°C in a yeast nitrogen base medium lacking leucine or uracil (Nagahora et al, 1992).…”

Antimicrobial activity of lectins from Antimicrobial Activity of Lectins from Plants

“…Expression of plant lectins was also achieved in other systems, e.g. WGA in Saccharomyces cerevisiae (Nagahora et al, 1992), PHA and GNA in Pichia pastoris (Raemaekers et al, 1999), PNA in insect cells (Kumar et al, 1999) and SBA in monkey cells (Adar et al, 1997); (for a more complete listing of recombinant plant lectins) (Streicher and Sharon, 2003).…”