目前以基因重組大腸桿菌生產異種蛋白質常見之醱酵策略，是以批次培養提高菌體濃度，再以饋料批次方式添加新鮮培養基與誘導劑異丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)；所以，過去的文獻大多以饋料的方式對於重組蛋白質生產之影響做為研究主題。然而到目前為止，並沒有研究人員深入討論直接影響蛋白質表現的IPTG之添加依據。有鑑於此本研究以Escherichia coli BL21 (DE3)/pET-43a為材料，探討白細胞介素20號(IL-20)的生產時，應根據何種原則添加IPTG。預先設定的IPTG之添加原則包括：(1)以固定濃度方式為之、(2)以誘導時的菌體量為依據、(3)以最終的菌體量為依據、(4)以誘導後菌體的增加量為依據。
研究結果顯示IL-20最終比產量與IPTG的添加量並無對應關係，但事實上重組蛋白質的最終比產量應該與IPTG添加量和菌體量都有關係，因此預先設定的四種原則均不適合做為IPTG之添加依據。然而，實驗數據卻顯示IL-20之比生產速率隨著IPTG添加濃度的增加而提高、誘導後第一小時內質體安定性與IPTG添加濃度成反比。除了IPTG的量以外，還有其他因素會造成重組蛋白質產量的差異，但如能以適當的IPTG濃度添加並配合養份的持續進料，可能會維持IL-20的生產。

The common strategy for producing recombinant proteins by Escherichia coli consists increasing cell density at the batch-cultured stage and feeding fresh medium with inducer at the fed-batch stage. For the past years, the effect of feeding strategy for the production of recombinant proteins had been studied extensively. However, the rule for adding inducer (isopropyl--D-thiogalactopranoside, IPTG), influences the production of foreign proteins, had never been discussed yet. Accordingly, the objective of this study was to investigate the rule of IPTG addition employing E. coli BL21 (DE3)/pET-43a as the IL-20 producing strain. The proposed rules for the addition of IPTG were according to: (1) constant IPTG concentration, (2) the cell mass at induction, (3) the cell mass at the end of fermentation, and (4) the increase of cell mass after induction.
The results first showed that there was no correlation between the amount of IPTG addition and the specific yield of IL-20. In fact, the specific yield of recombinant protein was affected by the amount of IPTG and cell mass. Therefore, the addition of IPTG can not determined by the rules proposed before. However, the data indicated that the specific production rate of IL-20 increased with the increase of IPTG concentration. Besides, the plasmid stability decreased in the first hour after induction as the increase of IPTG concentration. In addition to IPTG, the yield of recombinant protein was affected by other factors. However, it was proposed that the production of IL-20 might continue if both appropriate concentration of IPTG and fresh medium were fed to the fermentation system.

Aristidou, A. A., K. Y. San, and G. N. Bennett, “Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary carbon source,” Biotechnol. Prog., 15: 140-145 (1999).

Ball, E. H., “Quantitation of proteins by elution of coomassie brilliant blue R from stained bands after sodium dodecyl sulfate –polyacrylamide gel electrophoresis,” Anal. Biochem., 155: 23-27 (1986).

Blumberg, H., D. Conklin, W. F. Xu, A. Grossmann, T. Brender, S. Carollo, M. Eagan, D. Foster, B. A. Haldeman, A. Hammond, H. Haugen, L. Jelinek, J. D. Kelly, K. Madden, M. F. Maurer, J. Parrish-Novak, D. Prunkard, S. Sexson, C. Sprecher, K. Waggie, J. West, T.E. Whitmore, L. Yao, M. K. Kuechle, B. A. Dale, and Y. A. Chandrasekher, “Interleukin 20 : discovery, receptor identification, and role in epidermal function,” Cell, 104: 919 (2001).

Gombert, A. K., and B. V. Kilikian, “Recombinant gene expression in Escherichia coli cultivation using lactose as inducer,” J. Biotechnol., 60: 47-54 (1998).

Han, K., H. C. Lim, and J. Hong, “Acetic acid formation in Escherichia coli fermentation,” Biotechnol. Bioeng., 39: 663-671 (1992).

Han, K., J. Hong, and H. C. Lim, “Relieving effects of glycine and methionine from acetic acid inhibition in Escherichia coli fermentation,” Biotechnol. Bioeng., 41: 316-324 (1993).

Khlebnikov, A., and J. D. Keasling, “Effect of lacY expression on homogeneity of induction from the Ptac and Ptrc promoters by natural and synthetic inducers,” Biotechnol. Prog., 18: 672-674 (2002).

Kilikian, B. V., I. D. Suárez, C. W. Liria, and A. K. Gombert, “Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction,” Process Biochem., 35: 1019-1025 (2000).

Kim, C. H., J. Y. Lee, M. G. Kim, K. B. Song, J. W. Seo, B. H. Chung, S. J. Chang and S. K. Rhee, “Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase,” J. Ferment. Bioeng., 86: 391-394 (1998).

Koo, J., “Therapeutic options for psoriasis : review and update,” Saudi Med. J., 19: 232236 (1998).

Lebwohl, M., L. Drake, A. Menter, J. Koo, A. B. Gottlieb, M. Zanolli, M. Young, P. McClelland, “Consensus conference : Acitretin in combination with UVB or PUVA in the treatment of psoriasis,” J. Am. Acad. Dermatol., 45: 544-553 (2001).

Lee, J. H., Y. H. Choi, S. K. Kang, H. H. Park, and I. B. Kwon, “Production of human leukocyte interferon in Escherichia coli by control of growth rate in fed-batch fermentation,” Biotechnol. Lett., 10: 695-698 (1989).

Lee, S. Y., “High cell-density culture of Escherichia coli,” TIB., 14: 98-105 (1996).

Lee, S. Y., Y. K. Lee, and H. N. Chang, “Stimulatory effects of amino acids and oleic acid on poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli,” J. Ferment. Bioeng., 79: 177-180 (1995).

Lehninger, A. L., D. L. Nelson, and M. M. Cox, “Chapter 27 Regulation of Gene Expression,” Principles of Biochemistry, Worth, Inc., New York, p.941-983 (1993).

Luli, G. W., and W. R. Strohl, “Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations,” Appl. Environ. Microbiol., 56: 1004-1011 (1990).

Mendoza – Vega, O., E. Buri, and D. Speck, “Enhancement of recombinant cholera toxin B subunit production in Escherichia coli by applying a fed-batch control strategy,” Biotechnol. Lett., 17: 1037-1042 (1995).

Middelberg, A. P.J., “Preparative protein refolding,” TIB., 20: 437-443 (2002).

Miller, G. L., “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Anal. Chem., 31: 426-428 (1959).

Morten, R. S., and J. Glastrup, “Acetic acid and formic acid concentrations in the museum environment measured by SPME-GC/MS,” Atmos. Environ., 36: 3909-3916 (2002).

Neubauer, P., and K. Hofmann, “Efficient use of lactose for the lac promoter-controlled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions,” FEMS Microbiol. Rev., 14: 99-102 (1994).

Neubauer, P., K. Hofmann, O. Holst, B. Mattiasson, and P. Kruschke, “Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer,” Appl. Microbiol. Biotechnol., 36: 739-744 (1992).

Pan, J. G., J. S. Rhee, and J. M. Lebeault, “Physiological constraints in increasing biomass concentration of Escherichia coli B in fed-batch culture,” Biotechnol. Lett., 9: 98-94 (1987).

Phumathon, P., and G. M. Stephens, “Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture,” Enzyme Microb. Technol., 25: 810-819 (1999).

Rich, B. E., and T. S. Kupper, “Cytokines: IL-20 – a new effector in skin inflammation,” Curr. Biol., 11: R531R534 (2001).

Sadana, A., “Protein refolding and inactivation during bioseparation: bioprocessing implications,” Biotechnol. Bioeng., 48: 481-489 (1995).

Shiloach, J., J. Kaufman, A. S. Guillard, and R. Fass, “Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21(λDE3) and Escherichia coli JM109,” Biotechnol. Bioeng., 49: 421-428 (1996).

Wittmann, G., H. V. Langenhove, and J. Dewulf, “Determination of acetic acid in aqueous samples, by water-phase derivatisation, solid-phase microextraction and gas chromatography,” J. Chromatogr. A., 874: 225-234 (2000).

Wong, H. H., Y. C. Kim, S. Y. Lee, and H. N. Chang, “Effect of post-induction nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli,” Biotechnol. Bioeng., 60: 271-276 (1998).

Woyski, D., and J. R. Cupp-Vickery, “Enhanced expression of cytochrome P450s from lac-based plasmids using lactose as the inducer,” Arch. Biochem. Biophys., 388: 276-280 (2001).

Zhang, X., Z. Xia, B. Zhao, and P. Cen, “Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae,” Biotechnol. Lett., 24: 995-998 (2002).