Biotechnol. hog. 1983, 9, 122-127
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ARTICLES Production of Fusion Protein SpA::EcoRI in Batch Culture in a 60-L Airlift Tower Loop Reactor Lutz Brandes, Xiaoan Wu, Hans E. Maschke,+Heike Jurgens, Birgit Reinhardt,*and Karl Schugerl; Institut fiir Technische Chemie, Universitlit Hannover, Callinstrasse 3, D-3000 Hannover, Germany
Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60-L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum),40, or 42 "C. The following parameters were monitored: cell mass concentration (X),total cell counts (TCC), number of colony-forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), COZproduction rate (CPR), respiration quotient (RQ), and volumetric mass-transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased t o avoid oxygen-transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.
Introduction Recombinant protein production has increasing importance. Only a few investigations have dealt with problems important for production. The influence of the dilution ratehpecific growth rate on the plasmid copy number (Seo and Bailey, 1985, 19861, the steady state (Chang and Lim, 1987), cultivation and production in a two-stage reactor system (Park et al., 19891, and effect of acetic acid (Lee et al., 1988, 1989; Kracke-Helm et al., 1991) and amino acids (Mizutani et al., 1986) are some of these investigations. The aim of the present investigation was the characterization of the cultivation of Escherichia coli carrying three different multicopy plasmids and producing a fusion protein SpA::EcoRI where EcoRI decomposesthe DNA of the host, with on-line and off-line measurements to find relationships between process variables and process performance. Due to frequent off-line sampling and the large sample volumes used for off-line analysis of the cell properties, a rather large cultivation medium volume was necessary to avoid changes in the process conditions caused by sampling. In large stirred tank reactors, considerable inhomogeneity of the cultivation medium prevails. To simulate
* Author to whom correspondence should be addressed. + Genentech, Inc., 460 Point Bruno Boulevard, South San Francisco, CA 94080. 1 AKZO, IMMA (Institut fur Medizinische Membrananwendungen), bhder Strasae 28, 5600 Wuppertal 2, Germany.
8756-7938/93/3009-0122$04.00/0
this inhomogeneity in large reactors,a 60-L working volume airlift tower loop reactor was used, in which the dissolved oxygen concentration was nonuniform. It was expected that the nonuniformity of the temperature in the reactor would influence the temperature-shift-induced gene expression. Therefore, the temperature profile in the loop reactor was monitored during the growth phase, induction of the gene expression, and product formation.
Materials and Methods Strain and Plasmids. Escherichia coli JM103 (DSM, Braunschweig) with the following genotype was employed endA, A[lac,prol thi-l,strA,scbBlB,hsdR4,supE, A-, lF'traD36, proA+B+,lacIqZ, AM151. For more information, see Maschke (1991) and Maschke et al. (1992). The following plasmid combination was used: expression plasmid pMTC48 (4.8 kbp) constructed from pRIT32 and pRIF309+ (Figure 1)carrying the gene SpA:EcoRI under the transcriptional control of two independent promoters, P L ~ ~inducible u v ~ with isopropyl thiogalactoside (IPTG) and the thermally inducible A PRpromoter. In addition, it carries the ampicillin resistance gene. The repression plasmid pRK248 (9.2 kbp) overproduces the cI857 repressor protein and carries the tetracycline resistance gene. The protection plasmid pEcoR4 (6 kbp) encodes the DNA methylase of EcoRI and carries the chloramphenicol resistance gene (Figure 1). For more details, see Maschke (1991) and Maschke et al. (1992). Medium Composition and Precultures. LB (Luria
0 1993 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. hog.., 1993, Vol. 9, No. 2
123 EcoRl (0)
(fl pEcoR4 1929 bp
Hlnd Ill (1122)
BamHl (W)
P d I(lQ33)
Hlnd 111 (3207)
A Hlnd 111 (3201 Hlnd III'(780)
,
EcoRl(1166) PItl (1201) EcoRV (1267)
3938 bp
EcoRl (0) Xbal (4)
\
Pvul (2497)
,
Hlnd 111 (4014)
+
Xbal (919) plrouvs pMTC48
LcoRl(Q23) Pdl(962)
EcoRl(284) Pvul(2254)
PVUl(4322)~
H
i
n
d
l
l(914)l 0 Y
pRIF309+ Pat I(4196)
EcoRl (280.5)
Figure 1. Restriction maps of (A) pEcoR4 and (B) pMTC48 and its construction.
Bertani) medium consisting of 10 g L-' casein peptone, 5 g L-l NaC1, and 10 g L-' yeast extract (pH 7) was employed for the precultures. Only the preculture media contained antibiotica: 17 g L-' agar was autoclaved and along with 100 mg mL-' ampicillin (trisodium salt), 30 mg mL-' chloramphenicol (sulfate), and 10 mg mL-' tetracycline supplement was put into a Petri dish at 50-60 "C. The first and second precultures contained 75 mg mL-' ampicillin (trisodium salt), 20 mg mL-I chloramphenicol (sulfate), and 10 mg mL-I tetracycline. Microorganisms were transferred from the agar plate to the first preculture (20 or 40 mL of LB medium with antibiotics; 6-8 h of cultivation). The second preculture (600 mL or 2 L of LB medium with antibiotics; 12-16 h of cultivation) was inoculated with the first preculture. Finally, the second preculture was used for inoculation of an airlift tower loop reactor (ATLR) with a 60-L working volume using M9 minimal medium (no antibiotica supplement). The composition of the M9 minimal medium for the main culture is given in Table I. For more details, see Brandes (1991) and Wu (1992). Reactor. Cell cultivations were performed at 30 "C and pH 7 in a tower reactor of 2.3 m height and 0.2 m diameter with a draft tube of 1.8 m height and 0.15 m diameter and a working volume of 60 L. For more details, see Kracke-Helm et al. (1991). The reactor was equipped with an on-line aseptic medium sampling module (crossflow module, Waters) at the external loop and an in situ tubular filtration module (ABC Co., Puchheim) at the internal column. Analysis. To analyze the intracellular product, cells were lysed for 25 min with a 1ysozymeiETDA solution at
Table 1. Composition of the M9 Minimal Medium. 100 mL L-I 21 g L-I Na2HP04,25 g L-l KH2P04, salt soln 5 g L-l NaCl, 25 g L-l (NH4)2S04 1 mL L-' 0.1 M CaClz 1 mL L-I 1.0 M MgC12 100 mL L-' 100 g L-' glucose soln 0.2 mL L-' 5 mg L-I thiamin, 5 mg L-I biotin vitamin soln 1 mL L-l trace 1.38 g L-I ZnSO4.7H20, 5.40 g L-' FeC13.6H20, element soln 1.60 g L-l MnS04,0.17 g L-l CuC12, 0.56 g L-' CoS0
