本研究利用一個簡單的進料策略達到控制細胞的比生長速率。此進料策略連接一個濃縮基質儲槽及兩個混合槽，可以固定流速來模擬指數函數饋料。藉由培養Escherichia coli DH5α的研究證實，在饋料批式醱酵中，利用擬指數函數饋料方法可以使細胞的生長維持在預先設定的比生長速率。菌體以指數函數生長的時間長短與所控制的比生長速率大小有關；比生長速率愈小，可以操作的時間愈長。此饋料方法不僅操作方便，而且不需複雜的電腦控制技術和設備，它可以被廣泛地應用於饋料批式醱酵中，使菌體在任意設定的比生長速率下，生產外源蛋白質。

　　創傷弧菌是一株嗜鹽性革蘭氏陰性細菌，會對人類造成嚴重的傷口感染和致死性敗血症，所分泌到細胞間質的核酸分解酵素，是一種可以分解DNA及RNA的非特異性核酸分解酵素，此酵素具有優異的熱穩定性。本研究以基因重組大腸桿菌E. coli DH5α/pSI026醱酵生產此核酸分解酵素。在饋料批式醱酵之饋料策略上，利用擬指數函數饋料方法控制菌體的比生長速率，藉以得到核酸分解酵素生產之動力學參數。實驗結果發現，細胞內核酸分解酵素的產率係數 （單位為 U/g cell）隨著菌體比生長速率 的增加而減少；然而，酵素的比生產速率 （單位為 U/g cell-h）則隨著 的增加而增加，在 = 0.05 h-1下，可得到最大的 值。在饋料批式醱酵中，核酸分解酵素的生產僅與菌體比生長速率有關，而與營養物的饋料方法無關。在酵素的產能與生產速率的考量下，線性遞增的饋料方法不僅操作簡單，而且在饋料批式醱酵中非常適合基因重組大腸桿菌生產核酸分解酵素。
　　雖然文獻中已有許多提高重組蛋白質產量的研究，但是，到目前為止，相對地尚少有關於誘導劑添加策略的報導。為了釐清IPTG的誘導策略(包括添加量、添加時機、和添加方式)對重組蛋白質IL-20生產之影響，本研究是在控制菌體比生長速率的條件下，利用基因重組大腸桿菌E. coli BL21(DE3)/pET-43a，於饋料批式醱酵中生產IL-20。實驗結果發現，不同的誘導劑添加策略對於細胞的生長、質體穩定性、重組蛋白質IL-20的生產、內涵體的形成等，有相當顯著的影響。當IPTG以一次添加的方式誘導IL-20合成時，細胞內IL-20蛋白質的產率係數 (單位為mg IL-20/g cell)會隨著菌體比生長速率 而改變，在 = 0.1 h-1有一最大值。然而，當IPTG隨著營養物以連續饋料的方式誘導IL-20合成時卻發現， 與重組蛋白基因的表現無關。於饋料批式醱酵中，利用連續饋入IPTG的誘導策略，除了可以避免 的下降外，同時亦可以增加IL-20的產率；更可以維持較高的質體穩定性和形成較少含量的內涵體。

　　A simple feeding method for controlling specific growth rate in fed-batch culture was developed. With a concentrate reservoir and two mixing chambers in series, this method can use a constant feed rate to simulate the exponential feeding. Fed-batch cultures with Escherichia coli DH5αshowed that the present feeding method could sustain cell growth at predetermined specific growth rates, where the time length for exponential growth was dependent on the magnitude of the growth rate. The present feeding method is ease to operate, requires no computerized control equipments, and thus could expect an extensive application in fed-batch culture to produce heterologous proteins.
　　Vibrio vulnificus, a halophilic gram-negative bacterium, is associated with serious wound infections and fatal septicemia in human. The nuclease of V. vulnificus was produced in the periplasmic space, which was found to possess both DNase and RNase activities and to be thermally stable. This research use a recombinant strain, E. coli DH5α/pSI026, to produce V. vulnificus nuclease. The effect of specific growth rate m on the nuclease production was investigated in fed-batch cultures. The pseudo-exponential feeding method for controlling m was employed to obtain the desired data. It was found that the nuclease content of cells gP varied with m, with a maximum occurred at m = 0.05 h-1; however, the specific nuclease production rate qp increased with an increase in m. The nuclease production was dependent on m, irrespective of the feeding methods. 　When concerning the compromise between the nuclease yield and its production rate, the linear-gradient feeding method, being simple and adaptable, was adequate for the nuclease production.

　　Although the production of recombinant protein has been extensively investigated, very few studies have discussed strategies of inducer addition. For better understanding of the effect of IPTG inducing strategies on the Interleukin 20 (IL-20) production, a recombinant strain, E. coli BL21(DE3)/pET-43a, was used in this study under controlled specific growth rates to produce IL-20 at the fed-batch mode. The results revealed that different strategies of inducer addition affected cell growth, plasmid stability, IL-20 synthesis, and the formation of inclusion body. It was found that the IL-20 content in cells (gP) varied with when IPTG was added by pulse. In addition, the maximum gP was found to occur at = 0.1 h-1. However, the specific growth rate and recombinant gene expression was not correlated when IPTG was added continuously. The results also demonstrated that continuous IPTG feeding could prevent a decrease in specific growth rate, but could increase IL-20 yield; moreover, it could sustain higher plasmid stability and allow less inclusion body formation.

Ahrenholtz, I., M. G. Lorenz, and W. Wackernagel, “The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm,” Arch. Microbiol., 161: 176-183 (1994).

Asadullah, K., J. Eskdale, A. Wiese, G. Gallagher, M. Friedrich, and W. Sterry, “Interleukin-10 promoter polymorphism in psoriasis,” J. Invest. Dermatol., 116: 975-978 (2001a).

Asadullah, K., M. Friedrich, S. Hanneken, C. Rohrbach, H. Audring, A. Vergopoulos, M. Ebeling, W. D. Döcke, H. D. Volk, and W. Sterry, “Effects of systemic interleukin-10 on psoriatic skin lesions: histologic immunohistologic, and molecular biology findings,” J. Invest. Dermatol., 116: 721-727 (2001b).

Asadullah, K., H. D. Volk, and W. Sterry, “Novel immunotherapies for psoriasis,” TRENDS in immunology, 23: 47-53 (2002).

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).

Bech Jensen, E., and S. Carlsen, “Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts,” Biotechnol. Bioeng., 36: 1-11 (1990).

Benedik, M. J., and U. Strych, “Serratia marcescens and its extracellular nuclease,” FEMS Microbio. Letters, 165: 1-13 (1998).

Benjamin, E. R., and T. S. Kupper, “Cytokines: IL-20 – a new effector in skin inflammation,” Current. Biology., 11: R531-R534 (2001).

Bentley, W. E., N. Mirjalili, D. C. Andersen, and R. H. Davis, “Plasmid-encoded protein: the principle factor in the metabolic burden associated with recombinant bacteria,” Biotechnol. Bioeng., 35: 668-681 (1990).

Biedermann, K., H. Fiedler, B. S. Larsen, E. Riise, C. Emborg, and P. K. Jepsen, “Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli,” Appl. Environ. Microbiol., 56: 1833-1838 (1990).

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).

Bollag, D. M., and S. J. Edelstein, “Protein Methods,” Wiley-Less, 95-135 (1991).

Choi, J. H., K. J. Jeong, S. C. Kim, and S. Y. Lee, “Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence,” Appl. Microbiol. Biotechnol., 53: 640-645 (2000).

Corchero, J. L., and A. Villaverde, “Plasmid maintenance in Escherichia coli recombinant cultures is dramatically, steadily, and specifically influenced by features of the encoded proteins,” Biotechnol. Bioeng., 58: 625-632 (1998).

Curless, C. E., J. Pope, L. Loredo, and L. B. Tsai, “Effect of preinduction specific growth rate on secretion of granulocyte macrophage colony stimulating factor by Escherichia coli,” Biotechnol. Prog., 10: 467-471(1994).

Curless, C., J. Pope, and L. Tasi, “Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli,” Biotechnol. Prog., 6: 149-152 (1990).

Curless, C., K. Fu, R. Swank, A. Menjares, J. Fieschko, and L. Tsai, “Design and evaluation of a two-stage, cyclic, recombinant fermentation process,” Biotechnol. Bioeng., 38: 1082-1090 (1991).

Cutayar, J. M., and D. Poillon, “High cell density culture of E. coli in a fed-batch system with dissolved oxygen as substrate feed indicator,” Biotechnol. Lett., 11: 155-160 (1989).

Doelle, H. W., Ewing, K. N. and Hollywood, N. W. “Regulation of glucose metabolism in bacterial system,” Adv. Biochem. Eng., 23: 1-35 (1982).

Donovan, R. S., C. W. Robinson, and B. R. Glick, “Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter,” J. Ind. Microbiol., 16: 145-154 (1996).

Emborg, C., P. K. Jepsen, and K. Biedermann, “Two-level factorial screening of new plasmid/strain combinations for production of recombinant-DNA products,” Biotechnol. Bioeng., 33: 1393-1399 (1989).

Etana, P., A. Tamar, R. Avraham, B. S. Anat, and C. Amikam, “Biosynthesis of the lactose permease in Escherichia coli minicells and effect of carrier amplification on cell physiology,” J. Bio. Che., 258: 5666-5673 (1983).

Fomenkov, A., and S. Y. Xu, “Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease,” Gene, 163: 109-113 (1995).

Friedhoff, P., B. Kolmes, O. Gimadutdinow, W. Wende, K. L. Krause, and A. Pingoud, “Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis,” Nucleic Acids Research, 24: 2632-2639 (1996).

Fujimoto, M., A. Kuninaka, and H. Yoshino, “Identity of phosphodiesterase and phosphomonoesterase activities with nuclease P1 (a nuclease from Penicillium citrinum),” Agr. Boil. Chem., 38: 785-790 (1974).

George, G., “Optimizing the production of recombinant proteins in microorganisms,” AIChE J., 34: 1233-1248 (1988).

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 E. coli fermentation,” Biotechnol. Bioeng., 39: 663-671 (1992).

Heins, J., J. Suriano, H. Taniuchi, and C. Anfinsen, “Characterization of a nuclease produced by Staphylococcus aureus,” J. Bio. Chem., 242: 1016-1020 (1967).

Hellmuth, K., D. J. Korz, E. A. Sanders, and W. D. Deckwer, “Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1,” J. Biotechnol., 32: 289-298 (1994).

Hlavac, F., and E. Rouer, “Expression of the protein-tyrosine kinase p561ck by the pTRX vector yields a highly soluble protein recovered by mild sonication,” Protein Expr. Purif., 11: 227-232 (1997).

Iijima, S., S. Yamashita, K. Matsunaga, H. Miura, M. Morikawa, K. Shimizu, M. Matsubara, and T. Kobayashi, “Use of a novel turbidimeter to monitor microbial growth and control glucose concentration,” J. Chem. Tech. Biotechnol., 40: 203-213 (1987).

Jepsen, P. K., E. Riise, K. Biedermann, P. C. Roboz Kristensen, and C. Emborg, “Two-level factorial screening for influence of temperature, pH, and aeration on production of Serratia marcescens nuclease,” Appl. Environ. Microbiol., 53: 2593-2596 (1987).

Jobe, A., and S. Bourgeois, “lac Repressor-Operator interaction,” J. Mol. Biol., 69: 397-408 (1972).

Jones, I. M., S. B. Primrose, A. Robinson, and D. C. Ellwood, “Maintenance of some ColEl-type plasmids in chemostat culture,” Molec. Gen. Genet., 180: 579-584 (1980).

Khosla, C., J. E. Curtis, P. Bydalek, J. R. Swartz, and J. E. Bailey, “Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter,” Bio/Technology, 8: 554-558 (1990).

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).

Konstantinov, K., M. Kishimoto, T. Seki, and T. Yoshida, “A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli,” Biotechnol. Bioeng., 36: 750-758 (1990).

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

Korz, D. J., U. Rinas, K. Hellmuth, E. A. Sanders, and W. D. Deckwer, “Simple fed-batch technique for high cell density cultivation of Escherichia coli,” J. Biotechnol., 39: 59-65 (1995).

Kosinski, M. J., U. Rinas, and J. E. Bailey, “Isopropyl-β-D-thiogalactopyranoside influences the metabolism of Escherichia coli,” Appl. Microbiol. Biotechnol., 36: 782-784 (1992).

Kovacevic, S., L. E. Veal, H. M. Hsiung, and J. R. Miller, “Secretion of staphylococcal nuclease by Bacillus subtilis,” J. Bacteriol., 162: 521-528 (1985).

Kwon, Y. W., E. R. Jang, Y. M. Lee, Y. S. Kin, K. S. Kwon, H. S. Jang, C. K. Oh, and K. W. Kim, “Insulin-like Growth Factor‖induces interleukin- 6 expression via NFκB Activation in psoriasis,” Biochem. Biophys. Res. Commun., 278: 312-317 (2000).

Laemmli, U. K., “Cleavage of structural proteins during the assembly of the head of bacteriophage T,” Nature, 227: 680-685 (1970).

Lee, J., S. Y. Lee, and S. Park, “Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source,” Biotechnol. Technol., 11: 59-62 (1997).

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

Li, Y., J. Chen, Y. Y. Mao, S. Y. Lun, and Y. M. Koo, “Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli,” Process Biochem., 33: 709-714 (1998).

Linn, S., and I. R. Lehman, “An endonuclease from Neurospora crassa specific for polynucleotides lacking an ordered structure,” J. Bio. Chem., 240: 1287-1293 (1965).

Lilie, H., E. Schwarz, and R. Rudolph, “Advances in refolding of proteins produced in E. coli,” Curr. Opin. Biotechnol., 9: 497-501 (1998).

Luli, G. W., S. M. Schlasner, D. E. Ordaz, M. Mason, and W. R. Strohl, “An automatic, on-line glucose analyzer for feed-back control of fed-batch growth of Escherichia coli,” Biotechnol. Lett., 1: 225-230 (1987).

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).

Majewski, R. A., and M. M.Domach, “Simple constrained optimization view of acetate overflow in E. coli,” Biotechnol. Bioeng., 35: 732-738 (1990).

Mark, L., and A. Suad, “Treatment of psoriasis. Part 2. Systemic therapies,” J. Am. Acad. Dermatol., 45: 649-661 (2001).

Mark, L., D. Lynn, M. Alan, K. John, B. Alice, Z. Michael, Y. Melodie, and M. Patricia, “Consensus conference: Acitretin in combination with UVB or PUVA in the treatment of psoriasis,” J. Am. Acad. Dermatol., 45: 544-553 (2001).

Miao, F., and D. S. Kompala, “Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: l. batch cultures and kinetic modeling,” Biotechnol. Bioeng., 40: 787-796 (1992).

Michael, S., R. B. Kunnel, K. Navin, M. Sabine, and R. Ursula, “Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli,” J. Biotechnol., 68: 71-83 (1999).

Mignone, C. F., and C. A. Rossa, “Simple method for designing fed-batch cultures with linear gradient feed of nutrient,” Process Biochem., 28: 405-410 (1993).

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

Mitraki, A., and J. King, “Protein folding intermediates and inclusion body formation,” Bio/Technology, 7: 690-697 (1989).

Mori, H., T. Yano, T. Kobayashi, and S. Shimizu, “High density cultivation of biomass in fed-batch system with DO-stat,” J. Chem. Eng. Japan, 12: 313-319 (1979).

Nestle, M., and W. K. Roberts, “An extracelluar nuclease from Serratia marcescens I. purification and some properties of the enzyme,” J. Bio. Chem., 244: 5213-5218 (1969).

Okita, B., E. Arcuri, K. Turner, D. Sharr, B. D. Tito, J. Swanson, A. Shatzman, and D. Zabriskie, “Effect of induction temperature on the production of malaria antigens in recombinant E. coli,” Biotechnol. Bioeng., 34: 854-862 (1989).

Ott, R., and R. Kramer, “DNA hydrolysis by inorganic catalysts,” Appl. Microbiol. Biotechnol., 52: 761-767 (1999).

Paalme, T., K. Tiisma, A. Kahru, K. Vanatalu, and R. Vilu, “Glucose-limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate,” Biotechnol. Bioeng., 35: 312-319 (1990).

Ramesh, A., A. K. Panda, B. R. Maiti, and A. Mukhopadhyay, “Studies on plasmid stability and LTB production by recombinant Vibrio cholerae in batch and chemostat cultures: A lesson for optimizing conditions for chemical induction,” J. Biotechnol., 43: 45-51 (1995).

Reiling, H. E., H. Laurila, and A. Fiechter, “Mass culture of E. coli: medium development for low and high density cultivation of E. coli B/r in minimal and complex media,” J. Biotechnol., 2: 191-206 (1985).

Riesenberg, D., K. Menzel, W. A. Knorre, H. D. Pohl, D. Korz, E. A. Sanders, A. Roβ, and W. D. Deckwer, “High cell density fermentation of Escherichia coli expressing at controlled specific growth rate,” J. Biotechnol., 20: 17-28 (1991).

Riesenberg, D., and R. Guthke, “High-cell-density cultivation of microorganisms,” Appl. Microbiol. Biotechnol., 51: 422-430 (1999).

Robbins, J. W., and K. B. Taylor, “Optimization of Escherichia coli growth by controlled addition of glucose,” Biotechnol. Bioeng. 34: 1289-1294 (1989).

Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D. H. Hsu, R. Kastelein, K. W. Moore, and J. Banchereau, “Interleukin 10 is a potent growth and differentiation fator for activated human B lymphocytes,” Proc. Natl. Acad. Sci. USA., 89: 1890-1893 (1992).

Schein, C. H., and M. H. M. Noteborn, “Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature,” Bio/Technology, 6: 291-294 (1988).

Shin, C. S., M. S. Hong, D. Y. Kin, H. C. Shin, and J. Lee, “Growth-associated synthesis of recombinant human glucagons and human growth hormone in high-cell-density cultures of Escherichia col,” Appl. Micobiol. Biotechnol., 49: 364-370 (1998).

Srinivasan, V. R., M. B. Fleenor, R. J. and Summers, “Gradient-feed method of growing high cell density cultures of Cellulomonas in a bench-scale fermentor,” Biotechnol. Bioeng., 19: 153-155 (1977).

Suzuki, T., T. Yamane, and S. Shimizu, “Phenomenological background and some preliminary trials of automated substrate supply in pH-stat modal fed-batch culture using a setpoint of high limit,” J. Ferment. Bioeng., 69: 292-297 (1990).

Thompson, B. G., M. Kole, and D. F. Gerson, “Control of ammonium concentration in Escherichia coli fermentations,” Biotechnol. Bioeng., 27: 818-824 (1985).

Tsai, L. B., M. Mann, F. Morris, C. Rotgers, and D. Fenton, “The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell density Escherichia coli fermentation,” J. Ind. Microbiol., 2: 181-187 (1987).

Vogt, V. M., “Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae,” Eur. J. Biochem., 33: 192-200 (1973).

Vojtisek, V., and J. Slezak, “Penicillinamidohydrolase in Escherichia coli Catabolite Repression, Diauxie, Effect of cAMP and Nature of Enzyme Induction,” Folia. Microbiol., 20: 298-306 (1975).

Vyas, V. V., S. Gupta, and P. Sharma, “Stability of a recombinant shuttle plasmid in Bacillus subtillis and Escherichia coli,” Enzyme Microb. Technol., 16: 240-246 (1994).

Wang, H. Y., C. L.Cooney, and D. I. C.Wang, “Computer control of baker’s yeast production,” Biotechnol. Bioeng., 21: 975-995 (1979).

Wu, S. I., S. K. Lo, C. P. Shao, H. W. Tsai, and L. I. Hor, “Cloning and characterization of a periplasmic nuclease of vibrio vulnificus and its role in preventing uptake of foreign DNA,” Appl. Environ. Microbiol., 67: 82-88 (2001).

Yamada, K., “Bioengineering report. Recent advances in industrial fermentation in Japan,” Biotechnol. Bioeng., 19: 1563-1621 (1977).

Yamane, T., and S. Shimizu, “Fed-batch techniques in microbial processes,” Adv. Biochem. Eng., 3: 147-194 (1979).

Yasukawa, T., C. Kanei-Ishii, T. Maekawa, J. Fujimoto, T. Yamamoto, and S. Ishii, “Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin,” J. Biolog. Chem., 270: 25328—25331 (1995).

Yee, L., and H. W. Blanch, “Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli,” Bio/technology, 10: 1550-1556 (1992).

Yee, L., and H. W. Blanch, “Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli,” Biotechnol. Bioeng., 41: 781-790 (1993).

Yonemura, K., K. Matsumoto, and H. Maeda, “Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958,” J. Biochem., 93: 1287-1295 (1983).

Zabriskie, D. W., and E. J. Arcuri, “Factors influencing productivity of fermentations employing recombinant microorganisms,” Enzyme Microb. Technol., 8: 706-717 (1986).

Zabriskie, D. W., D. A. Wareheim, and M. J. Polansky, “Effects of fermentation feeding strategies prior to induction of expression of a recombinatn malaria antigen in Escherichia coli,” J. Ind. Microbiol., 2: 87-95 (1987).