Abstract
Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.
Highlights
Owing to its generally recognized as safe (GRAS) status, the yeast Saccharomyces cerevisiae has been widely used as a popular workhorse for the production of various pharmaceutical and industrial proteins
Voa1p consists of 265 amino acids containing a secretion signal peptide, three glycosylation sites, a proposed hydrophilic domain (HL), and a transmembrane domain (TM) (Fig. 1a)
A generally preferred method for enhanced secretion and efficient purification of recombinant proteins is to express them with fusion partners
Summary
Owing to its generally recognized as safe (GRAS) status, the yeast Saccharomyces cerevisiae has been widely used as a popular workhorse for the production of various pharmaceutical and industrial proteins. The yeast expression system combines the advantages of both bacteria and higher eukaryotic cells (Kunes et al 1987). The yeast protein secretion pathway and posttranslational modification systems are similar to those of higher eukaryotic cells, the fidelity of protein hyperglycosylation varies according to the specific target (Buckholz and Gleeson 1991). Yeast can produce various complex proteins that are native to higher eukaryotes including humans; this is a distinct advantage over production in Escherichia coli, as proteins lack eukaryotic modifications and are often inactive. Secretory production of foreign proteins greatly simplifies the purification procedure and reduces the production cost compared to intracellular production
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