TM Yeast Pst1 Recombinant Protein

Protoplast secreted protein 1 (Pst1) is an anchored plasma membrane protein. Pst1 was previously identified as a protein secreted by yeast regenerating protoplasts, which suggests a role in cell wall construction (1) . Pst1 is a protein with 444 amino acids and is attached to yeast cell wall via a glycosylphosphatidylinisotol (GPI) anchor (2) . Pst1 contains 15 potential N-linked glycosylation sites and is heavily glycosylated. It migrates at around 200 kDa on SDS-PAGE when produced in wild type S. cerevisiae. Our Pst1 protein was expressed in a genetically manipulated triple-mutant (TM) S. cerevisiae stain (Δoch1 Δmnn1 Δmnn4), which results in the production of sole Man8GlcNAc2 carbohydrate structures and Pst1 migration at approximately 100 kDa. When produced in the TM yeast, Pst1 can be recognized by several HIV-1 broadly neutralizing antibodies, including 2G12 and recently identified glycan-specific PGT antibodies (3) . Among several heavily N-glycosylated TM yeast glycoproteins, Pst1 shows high affinity for 2G12 and efficiently inhibits gp120 interactions with 2G12 and DC-SIGN, and it also blocks 2G12-mediated neutralization of HIV-1 pseudoviruses (4) .

Store in working aliquots at -70˚ C. Avoid freeze/thaw cycles. When working with proteins care should be taken to keep recombinant protein at a cool and stable temperature.

Pardo, M, Monteoliva L, Pla J, et al., Two-dimensional analysis of proteins secreted by Saccharomyces cerevisiae regenerating protoplasts: a novel approach to study the cell wall. Yeast. 1999 Apr;15 (6) :459-72.

Pardo, M, Monteoliva L, Vázquez P, et al., PST1 and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity. Microbiology. 2004 Dec;150 (12) :4157-70.

Luallen, RJ, Lin JQ, Fu H, et al., An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies. J Virol. 2008 July; 82 (13) : 6447-6457.

Luallen RJ, Fu H, Agrawal-Gamse C, et al., A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN. J Virol. 2009 May; 83 (10) : 4861-4870.