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Expression of a functional human interleukin-18 in yeast

Expression of a functional human interleukin-18 in yeast,10.1016/S0141-0229(02)00043-1,Enzyme and Microbial Technology,Young-Yi Lim,Mi-Yae Lee,Bong-Wo

Expression of a functional human interleukin-18 in yeast   (Citations: 5)
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The cDNA sequence for mature human interleukin-18 gene (hIL-18) was cloned and then used to transform Saccharomyces cerevisiae. Two different promoters for heterologous expression of hIL-18 were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Northern blot analysis revealed that, although variation in the expression level of rhIL-18 existed among transformants, the highest expression was obtained by the GPD promoter. Expressed hIL-18 protein (rhIL-18) was successfully secreted into culture medium due to the presence of the signal peptide of rice amylase 1A. It was possible to produce 13 mg of rhIL-18 protein per liter of culture filtrate without any changes in cell growth. Both cell growth and rhIL-18 production reached the peaks after the 3-day cultivation while the accumulation of transgene transcript peaked at 24 h of cultivation. The secreted rhIL-18 had an estimated molecular mass of 18 kDa. The bioassay observing the induction of interferon-γ from the KG-1 cell line indicated that the secreted recombinant rhIL-18 was bioactive and the specific activity of yeast-derived rhIL-18 was enhanced 15 times relative to that of E. coli-derived rhIL-18.
Journal: Enzyme and Microbial Technology - ENZYME MICROB TECHNOL , vol. 30, no. 6, pp. 703-709, 2002
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    • ...A wide variation in heterologous gene expression in S. cerevisiae is not unusual when episomal 2 μ ori-based plasmids are used, possibly because of variation in the plasmid copy number between different transformants [33,34]...
    • ...Whether because of protease activity or other unknown reasons, the instability of laccase seems to be specific rather than general, because the same host strain is used for the expression of various proteins, including enzymes, cytokines, and structural proteins [33,35,44], without noticeable instability of expressed target proteins...
    • ...The signal peptide (ASP) of amylase 1A (Ramy1A) from rice, which has been used in several previous studies to obtain high levels of secretion [33,34], was fused to a mature peptide of the laccase3 protein via overlap extension PCR to create XbaI restriction sites (italics) at the 5’ and 3’ ends using the following primers: forward 5’-GGTCTAGAATG CAGGTGCTGAACACC-3’ and reverse 5’-GGTCTA-GATTATATGCCC GAATC GTCC-3’, and overlap-forward ...

    Jung-Mi Kimet al. Heterologous expression of a tannic acid-inducible laccase3 of Cryphon...

    • ...The growth curve of TYEGLTB-4 in the YEPD broth exhibited canonical lag, log, deceleration, stationary, and decline phases (Fig. 3). The growth pattern of the selected transformant was similar to that of the control strain, and appeared to be similar to those determined in other previous studies [24, 30, 38]...
    • ...These early peaks and rapid declines for both the recombinant and endogenous gene transcripts derived from the GPD promoter were also demonstrated in previous studies [24] and appeared to reflect the characteristics of the GPD promoter under the present culture conditions...
    • ...Again, the titration effect induced by the dilution of the necessary transcription factors resulting from the presence of a multicopy promoter may explain the observed rapid decline of GPD and LTB expressions in the transformants [24]...

    Jung-Gu Limet al. Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit ...

    • ...The pMYO53 plasmid containing the codon-optimized synthetic gene encoding cholera toxin B subunit (CTB) [24] and prhIL-18 plasmid containing the amylase 1A (Ramy1A) signal peptide (ASP) [26] were used as templates for PCR amplifications of CTB and ASP genes, respectively...
    • ... from the recipient strain (Fig. 2). However, the level of accumulation of the recombinant CTB transcripts varied between strains transformed with the same expression construct (Fig. 2). Several studies have also shown a wide variation in the expression level of heterologous genes in S. cerevisiae when using episomal 2 μ oribased plasmids, possibly due to the variations in the plasmid copy number between different transformants [ ...

    Jung-Gu Limet al. Heterologous expression of cholera toxin B subunit in Saccharomyces ce...

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