A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae

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.

Keywords: Fusion partner; Purification; Saccharomyces cerevisiae; VOA1.

Fig. 1

Expression analysis of C-terminally truncated VOA1 derivatives. a The predicted amino sequence and domains encoded by the VOA1 gene. Truncation sites are indicated by arrows. Glycosylation sites are highlighted in bold. b Schematic diagram of serially deleted VOA1 genes. Sig signal peptide, N N-linked glycosylation site, HL hydrophilic domain, TM: transmembrane domain. c Expression levels of VOA1 derivatives were analyzed by SDS-PAGE. Lanes 1–5 culture broth of S. cerevisiae transformed with YGaT41–YGaT45 vector, respectively; lane M pre-stained protein size marker (Invitrogen)

Fig. 2

Expression analysis of VOA1 derivatives fused with hIL2. a Schematic representation of VOA1-hIL2 fusion proteins. Sig signal peptide, N N-linked glycosylation site, HL hydrophilic domain. b Confirmation of hIL2 expression by SDS-PAGE analysis. Lanes 1–3 culture broth of S. cerevisiae transformed with YGaT42-IL2, YGaT43-IL2, and YGaT44-IL2 vector, respectively; lane M pre-stained protein size marker (Invitrogen). c Western blot analysis of intracellular proteins. Lanes 1–3 cell extract of recombinant strains carrying YGaT42-IL2, YGaT43-IL2, and YGaT44-IL2 vector, respectively

Fig. 3

Effects of HL50 tagging on the expression of hEGF. a Schematic representation of the hEGF secretion cassette. b SDS-PAGE analysis of culture broth of S. cerevisiae transformed with YEGα-EGF (lane 1), YEGα-HL50-EGF (lane 2), and YEGα-EGF-HL50 (lane 3), respectively; lane M pre-stained protein size marker (Invitrogen)

Fig. 4

Expression of hEGF using modified HL peptides. a Amino acid sequence of modified HL peptides. b Schematic representation of the hEGF secretion cassette. c SDS-PAGE analysis of culture broth of S. cerevisiae transformed with YEGα-EGF (lanes 1, 5, 9); YEGα-HL50-EGF (lanes 2, 6, 10); YEGα-HL37-EGF (lanes 3, 7, 11); and YEGα-HL28-EGF (lanes 4, 8, 12); respectively. Lanes 5–8 after deglycosylation, lanes 9–12 after digestion with EK, lane M pre-stained protein size marker (Invitrogen)

Fig. 5

Confirmation of recombinant hEGF expression. a Time profiles of fed-batch fermentation of S. cerevisiae expressing YEGα-HL28-EGF. b SDS-PAGE analysis of culture supernatants. Samples of culture supernatants (10 μl) at the indicated times were analyzed. c SDS-PAGE of the purified hEGF. Lane 1 after ultrafiltration of fermentation broth, lane 2 after Ni-NTA affinity chromatography, and lane 3 purified hEGF after EK digestion. d Bioactivity assay of the purified hEGF. The EL-4 cell line was cultured in the presence of the indicated amounts of hEGF, and cell proliferation was analyzed following bromodeoxyuridine (BrdU) labeling

Fig. 6

Expression of EXD-4 and hIGF-1 using HL28. SDS-PAGE analysis of culture broth of S. cerevisiae transformed with YEGα-EXD4 (lane 1), YEGα-HL28-EXD4 (lane 2), YEGα-IGF (lane 3), and YEGα-HL28-IGF (lane 4), lane M pre-stained protein size marker (Invitrogen)