Abstract
To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins.
Highlights
Recombinant production of valuable proteins that are expressed in limited amounts in nature is essential for generation of human biopharmaceuticals and industrial biocatalysts
We found that invertase exhibited different fates when it was directly fused to human serum albumin (HSA) or human interleukin (hIL)-2
This feature of invertase fused with a low-secretion-competent protein (X) like hIL-2 made it possible to design a genetic trap for target protein-specific translational fusion partner (TFP) from a fusion partner library derived from truncated cDNAs and genomic DNA (Fig. 1b)
Summary
Recombinant production of valuable proteins that are expressed in limited amounts in nature is essential for generation of human biopharmaceuticals and industrial biocatalysts. S. cerevisiae has a secretion pathway and post-translational modification system, similar to those of higher eukaryotes, except the possibility of hyperglycosylation of the recombinant proteins It can produce various complex proteins originating from higher eukaryotes at low cost, which are often expressed in inactive forms as inclusion bodies in E. coli. Trans-acting chaperones[11,12] and cis-acting chaperones (secretory fusion partners)[13,14,15], have been used to enhance protein folding rates and avoid aggregation and degradation Such trans- and cis-acting chaperones have been used to improve the secretion efficiency of some proteins, many negative examples, often unpublished, have been reported for other proteins with the same chaperones[16,17,18]. High-throughput, positive selection of a target protein-specific translational fusion partner (TFP) harbouring a secretion signal and fusion partner from truncated genomic and cDNA libraries could be performed by simple selection of cells growing on sucrose medium. This method could be used to identify various secretion signals and fusion partners from the TFP library for the secretion of individual proteins
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