JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 82, No. 2, 128-133. 1996

An Approach to High-Level Production of a Mecasermin (Somatomedin C) Fused Protein in

Escherichih cob HBlOl YUJI NOGUCHI, SUSUMU SATOH, MICHITAKA YAMAGUCHI, KATSUYUKI WATANABE,

MASAKO HAYASHI, HISASHI YAMADA, YOSHIMASA SAITO,* MASAKAZU KOBAYASHI, AND KYOICHI SHIMOMURA

Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Yodogawa-ku, Osaka 532, Japan

Received 19 March 1996/Accepted 9 May 1996

A method to produce a mecasermin fused protein on an industrial scale was investigated. We constructed a new expression vector (designated pLSD1) derived from pLHSdMmtrp (Saito, Y. et al.: J. Biochem., 101, 123-134, 1987) by introduction of a synthetic fd phage terminator and deletion of the rop region originating from pBR322. The plasmid, pLSD1, exhibited high stability and was present at high copy number in Escherichia coli HBlOl. Although the growth of E. coli HBlOl/pLSDl was not suficient for high-level production of the fused protein in a Trp-deficient medium such as MBCA medium, it was improved by growing the strain in a medium containing 0.7% yeast extract which was constantly supplied with glucose. E. coli HBlOl requires Pro and Leu for its growth; however, excess Leu tended to inhibit the cell growth. From the results of investigation of the mechanism of inhibition by Leu, addition of Ile was found to prevent the inhibition. Consequently, high-level production of the fused protein was attained by (i) using pLSD1 as the expression vector, (ii) culti- vation of E. coli HBlOl/pLSDl in a medium containing 0.7% yeast extract, (iii) fed-batch cultivation with periodic addition of glucose, and (iv) addition of Pro, Leu and Be during cultivation. The cell density reached an OD at 600 nm of 50.4 and production of the fused protein was 1.24 g/l broth in a 150 1 fermentor.

[Key words: mecasermin, somatomedin C, fed-batch cultivation, Escherichia coliHBlO1, recombinant DNA]

Mecasermin, also called somatomedin C or insulin-like growth factor-I (IGF-I), is a 70-amino acid polypeptide hormone found in human plasma (1). It functions in the incorporation of sulfates into bone cells and stimulates proliferation of fibroblasts and cartilage cells (2). The growth promoting effect of somatotropin is mediated by mecasermin which is released from various types of cells stimulated with somatotropin (3). Mecasermin is useful for Laron-type dwarfism (4) and somatotropin insensitivi- ty syndromes (5). It also shows insulin-like hypoglycemic activity via a pathway independent of that of insulin. Therefore it is effective for treatment of diabetic patients with abnormal insulin receptors (6, 7). It might also be useful in the treatment of osteoporosis, skin burns and bleeding ulcers (8). To obtain the quantities of mecaser- min necessary to treat patients effectively, various ap- proaches for production of mecasermin involving recom- binant DNA techniques have been used (g-13), because the amount of mecasermin which can be isolated from natural sources is severely limited (1, 2). We have also reported on the production of mecasermin fused to a portion of interferon-r using recombinant Escherichia coli (14). For production of mecasermin on an industrial scale, E. coli HBlOl was selected as the host strain, because it requires Leu and Pro for its growth and there- fore conforms to biosafety regulations. Shimizu et al. reported mass production of recombinant epidermal growth factor in E. coli HBlOl by continuous addition, as nitrogen sources, of yeast extract, casamino acids and Trp in the growing phase and then casamino acids only in the expression phase (15). However, we aimed to per- form high cell density cultivation of E. coli HBlOl by

* Corresponding author.

supplying particular amino acids only. In this paper, we describe the approach we used to achieve large scale production of the mecasermin fused protein by improv- ing the expression vector and optimizing the cultivation method of the recombinant E. coli HBlOl.

MATERIALS AND METHODS

E. coli strain and materials E. coli HBlOl [F-, hsd20 (rB_, mB_), recA13, ara-14, proA2, leu-, IacYl, galK2, rspL20 (Srrf), ~~1-5, mtl-I, supE44, 2-1 was used as a host strain for expression experiments. Restriction and modification enzymes were purchased from Takara Shuzo (Kyoto), Toyobo (Osaka) and New England Biolabs (Beverly, USA). Amino acids were obtained from Kyowa Hakko (Tokyo), yeast extract was from Difco (Detroit, USA), and porcine insulin was from Sigma (Milwaukee, USA).

Genetic engineering techniques pLHSdMmtrp car- rying a DNA fragment coding for mecasermin fused to a portion of interferon-i (14, 16) and pCLaHtrp3t car- rying an fd phage central terminator (17) were used as starting plasmids. All DNA manipulations were per- formed according to methods described by Sambrook et al. (18). DNA sequencing was performed using the Maxam-Gilbert method (19) and a 370A DNA sequencer (Applied Biosystems, Foster City, USA).

Large scale cultivation of E. coli E. coli HBlOl/ pLSD1 stored in 20°d glycerol (l.Oml) at -190°C was cultivated in 100 ml of LB broth containing 50 pg/ ml of ampicillin at 37°C for 8 h. The broth was trans- ferred to 141 of a 0.4.9; yeast extract medium [0.4,0,’ yeast extract, 0.7,%’ Na2HPOJ. 12H20, 0.49; KH2P04, 0.4Pd K2HP04, O.lZ,?d (NH&SO‘,, 0.02,“d NH&l,

128

VOL. 82. 1996 HIGH-LEVEL PRODUCTION OF SOMATOMEDIN C 129

0.012% MgS04. 7H20, 0.00012% FeSO., . 7H20, 0.000012% CaC12.2H20, 0.00003% MnS0,.nH20, 0.00003% AlC&. 6H20, 0.000012% CoC13.6H20, 0.000006% ZnSO,. 7H20, 0.000006% NazMo04. 2H20, 0.000003% CuS04.5H20, 0.0000015% H3B03, pH 7.01 containing 0.5% glucose and 50 pg/ml ampicillin in a 20 I fermentor and the mixture was cultivated at 37°C under stirring at 300 rpm with aeration at 7.0l/min for 16 h. The cultivated broth was transferred to 761 of a 0.7% yeast extract medium [0.7% yeast extract, 0.7% Na2HP04. 12H20, 0.7% KH2P04, 0.7% K2HP04, 0.12% (NH&SG4, 0.02% NH&l, 0.11% MgS04.7Hz0, 0.0011% FeS04.7Hz0, 0.0011% CaC12.2H20, 0.003% MnS04. nH*O, 0.0003% A1C13. 6H20, 0.00012% CoCl,. 6Hz0, 0.00006% ZnS03. 7Hz0, 0.00006% Na2Mo04. 2HZ0, 0.00003% CuSO‘,. 5H20, 0.000015% H3B03, pH 7.01 containing 0.5% glucose, 25 pg/ml ampicillin and each 0.155% of Leu, Ile and Pro in a 150 I fermen- tor. The starting cultivation conditions were stirring at 300 rpm and aeration at 50 I/min under 0.5 kg/cm* pres- sure at 37°C and the conditions were gradually changed to stirring at 400 rpm and aeration at 140Vmin under 1.4 kg/cm2 pressure at 35°C to maintain the required oxygen level. The pH of the broth was maintained at 7.0 with 28% aqueous NH40H. To promote cell growth, 0.5% glucose (final concentration) was added every 30min after 1.5 h, 1.0% glucose after 7 h and 0.5% glu- cose after 9 h [2 times]. To avoid glucose suppression on cell growth, the concentration of glucose was maintained at less than 2% with monitoring with F-Kit o-glucose (Boehringer Mannheim, Mannheim, Germany). Leu, Pro and Ile (0.04% each, final concentration) were added to the culture medium at 7 and 7.5 h. For induction of promoter activity, 10 pg/ml ,3-indoleacrylic acid was added every 30min for a total of 9 times after an OD at 600 nm of 10 was reached. The cells were harvested when an OD at 600nm of 50.4 was reached. The yield of wet cells was 10.7 kg from 108 I (final volume).

HPLC analysis The cells from 20ml of the culture broth were suspended in 15 ml of 10mM PBS (pH 8.0) containing 83 mM EDTA and treated with 1.0 mg/ml lysozyme (Seikagaku Co., Tokyo) at ambient tempera- ture for 60 min. After addition of 20 ml 2% Sarcosyl in 25 mM PBS the reaction mixture was sonicated at 4°C and centrifuged (4’C, 15,00Orpm, 30min) to give fused protein. The fused protein was suspended in 0.1 M Tris-HCl (pH 7.0) containing 6 M guanidine-HCl, 10mM EDTA and 10% 2-mercaptoethanol, lysed by sonication and centrifuged. The supernatant was filtered through a 0.45 pm membrane filter (Millipore, Tokyo) and subjected to gel-filtration HPLC [column, TSKGel 3OOOSWxL (7.8 mm+ X 30 cm, Tohso, Tokyo); eluate, 0.1 M Tris-HCl (pH 7.0) containing 6 M guanidine-HCl, 10mM EDTA and 10mM DTT; flow rate, 0.5 ml/min; detection wavelength, 280 nm]. The fraction correspond- ing to the main peak eluted at approximately 18 min was collected and analyzed by reversed-phase HPLC [column, YMC ODS AP-320 (4.6mm$ x 15 cm, YMC, Uji); eluate, a linear gradient from 25% to 60% acetoni- trile in 0.1% trifluoroacetic acid over 30 min; flow rate, 1 .O ml/min; detection wavelength, 214 nm]. The concen- tration of the protein was calculated by comparing the peak area with that of porcine insulin analyzed by the reversed-phase HPLC under the same conditions.

Protein chemistry Amino terminal sequencing was performed using a 470A protein sequencer (Applied

Biosystems, Foster city, USA), and amino acid composi- tion was determined using an amino acid analyzer (835, Hitachi, Tokyo).

RESULTS AND DISCUSSION

Construction of an expression vector It has been reported that stable, high copy number of expression vectors are essential for high-level expression of recom- binant protein in E. cob. Therefore, we prepared a new expression vector, designated pLSD1 (Fig. 1) from pLHSdMmtrp (14) by introduction of an fd phage cen- tral terminator (17) and deletion of the rop region originating from pBR322. The plasmid pLSD1 in E. co/i HBlOl was extremely stable; no plasmid loss was ob- served during cultivation in a tryptophan-deficient M9CA broth for 48 h. The copy number of pBR322 is controlled by the rop region (18). The deletion of the rop region resulted in the copy number of pLSD1 being twice that of pLHSdMmtrp. Similar results were ob- tained by Twigg and Sherratt (20) during construction of pAT153. Consequently, expression of the fused protein from pLSD1 in M9CA broth increased to 1.7 times that from pLHSdMmtrp (data not shown). Therefore, we used E. coli HBlOl/pLSDl to investigate the conditions for attaining high-level expression of the mecasermin fused protein.

Characterization of the fused protein E. coli HBlOl/pLSDl was cultivated in a 0.7% yeast extract medium and the cellular lysates were analyzed by SDS- PAGE (Fig. 2). The mecasermin fused protein (detected as a 15 kDa protein) was expressed at a high level (lane 1) and was recovered in PBS-insoluble proteins (lane 3). No band corresponding to the fused protein was found in PBS-soluble extracts (lane 2). Therefore, the fused protein was easily purified by treating the cells succes- sively with lysozyme and detergents (lane 4). The fused protein thus obtained was sufficiently pure for the isola-

Hpd 70

L ApaLI 1300

FIG. 1. Structure of pLSD1, an expression vector for mecasermin fused protein. pLHSdMmtrp (14) was digested with BumHI and Sal1 and ligated to the 47-bp BumHI-Sun DNA fragment encoding thefd phage central terminator of pCLaHtrp3t (16). The resulting plasmid was digested with PvuII and &/I, and the cohesive ends were filled-in with DNA polymerase I (Klenow fragment) and self-ligated to give pLSD1. trp: E. coli tryptophan promoter; LH-SMC: mecasermin fused to a portion of interferon-y [Cys’-Leu59, numbers correspond to those by Gray et a/. (2O)I; Amp: ampicillin resistance gene; ter: fd phage central terminator; Ori: origin of replication.

130 NOGUCHI ET AL. J. FERMENT. BIOENG..

Ml 2 34

FIG. 2. SDS-PAGE analysis. E. coli HBlOl/pLSDl was culti- vated in a 0.7% yeast extract medium. The cellular lysates were ana- lyzed by 20% SDS-PAGE. Lane M, molecular weight markers; from the top upper, 94,000, 67,000, 43,000, 30,000, 21,000, 14,400; lane 1, total cellular lysate obtained by sonication of cells in PBS containing 8 M urea; lane 2, cellular PBS-soluble fraction; lane 3, cellular PBS- insoluble fraction (re-extracted with PBS containing 8 M urea); lane 4, isolated fused protein.

tion of mecasermin by limited cyanogen bromide degrada- tion (described in a subsequent paper).

The amount of the fused protein expressed was deter- mined using a combination of gel-filtration and reversed- phase HPLC (Fig. 3a, b). The structure of the fused protein was confirmed by amino terminal sequencing as well as amino acid analysis (see the legend for Fig. 3).

Effect of Trp level on the growth of E. coli HBlOl Although approximately 20mg/l of the fused protein was expressed in E. coli HBlOl/pLSDl cultivated in M9CA broth, this level of production was still unsatisfac- tory for large scale production of mecasermin. The low level of expression in M9CA broth seemed to be due to the limited cell growth in this medium (OD at 600 nm of 1.6 in the stationary phase). Trp-deficient medium, such as MBCA broth, has been generally used for expression of genes under the control of the trp promoter, carried on plasmids harbored by E.coli (21, 22). However, the growth of recombinant E. coli tends to be inhibited in the early growth phase in this type of medium due to the stress of expressing the foreign protein (23). To over- come these problems, we investigated various types of culture media that contain moderate amounts of Trp, and found that a medium containing 0.7% yeast extract supplemented with phosphates and metal ions was optimal for cell growth as well as protein expression. The concentration of Trp in the medium was 35.0t 7.9 [(g/ml at the start of cultivation, then decreased to less than 10 /‘g/ml during incubation at 37°C for 4.5 h at which point the level of Trp was sufficiently low for the induction by ,3-indoleacrylic acid (see also Materials and Methods).

Effects of Pro and Leu on the growth of E. coli HBlOl Mori et al. (24) have reported the conditions required for high density cultivation of E. coli B in a 0.4:; yeast extract medium with continuous addition of

10 20

Time (min)

3

0 IO 20 30 Time (min)

FIG. 3. HPLC analysis of mecasermin fused protein. HPLC conditions are described in Materials and Methods. (a) Gel-filtration HPLC chromatogram. The semi-purified fused protein was analyzed using a TSKGel 3OOOSWxL column. The fractions shown by arrows were collected and analyzed by reversed-phase HPLC. (b) Reversed- phase HPLC chromatogram. The collected fraction was analyzed using a YMC ODS AP-320 column. The peak eluted at approximately 24min was collected and used for amino terminal and amino acid composition analyses. The arrow indicates fused protein.

glucose. Using their methods, we cultivated E. coli K12 and obtained a similar result with them (data not shown). However, the growth of E. coli HBlOl/pLSDl in the same medium ceased at 4 h after the start of culti- vation when an OD at 600nm of 4.6 was reached (Fig. 4). Although the growth resumed on addition of 0.4% yeast extract (final concentration) (solid arrow in Fig. 4), it ceased again at 6.5 h. Then, 50 pg/ml each of Leu and Pro (final concentration) were added twice at 7 and 8 h to complement the nitrogen source (dashed arrows in Fig. 4) since E. coli HBlOl requires Pro and Leu for its growth. The OD at 600nm increased to 12.6 at 8.5 h. In the experiment, the glucose level was controlled to less than 2%. From these data, the increase in DO level seemed to correspond to the deficiency of the nitrogen source in the broth (Fig. 4). We tried to control the DO level in the broth containing 0.7,00/ yeast extract which was effective for both cell growth and protein expres- sion. Then, we cultivated E. coli HBlOl/pLSDl in 0.7,00’ yeast extract medium (90 r) in a 150 I fermentor with suc- cessive addition of Pro and Leu (Fig. 5). Pro and Leu (final concentration 50 /ig/ml each) were added periodi- cally from O-5 h after the start of the cultivation (a total of 6 times). After 5.5 h, these amino acids were added at the times when the DO level increased (a total of 9

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8 g 60 40: :

20 -

Time (h)

FIG. 4. Cultivation of E. cob HBlOl/pLSDl with addition of nitrogen sources. E. coli HBlOl/pLSDl was cultivated in a 5 I fermentor containing 0.4% yeast extract medium. Arrows indicate the time when nitrogen sources were added. Solid arrow, 0.4% yeast extract; dashed arrows, 50pg/ml each of Leu and Pro (final con- centration).

times). The addition of Pro and Leu resulted in an in- crease in the final OD at 600 nm (=26.4) as compared to that obtained from the conditions of Fig. 4. The expres- sion of the fused protein was also improved (715 mg/l).

1

7-----Tooo

,110 0 2 4 6 a 10

Time (h)

5. Cultivation of E. coli HBlOl/pLSDl with successive addition of Leu and Pro. The E. co/i was cultivated in 0.7% yeast ex- tract medium in a 150 I fermentor. Both Leu and Pro (at a final con- centration of 50 pg/ml each) were added during the cultivation at the times shown as arrows. Open circles, OD; closed circles, the fused protein.

HIGH-LEVEL PRODUCTION OF SOMATOMEDIN C 131

TABLE 1. Concentration of Pro and Leu during cultivation

Time Pro Leu (h) (mg/l) (mg/0

0 157 286 4.5 66.2 94 7.0 10.2 5.5

Cultivation conditions are described in the legend of Fig. 5.

However, the concentrations of Leu and Pro in the fer- mentation broth remained low in spite of successive addi- tion of these amino acids (Table 1). This indicates that Pro and Leu are consumed rapidly at a rate proportion- al to that of cell growth. On the other hand, cultivation of E. coli with periodical addition of Pro and Leu (50 pg/ml each) at the same time as glucose was fed was less effective for expression of the fused protein (410mg/l). These results indicate that amino acids essen- tial for the growth of E. coli HBlOl should be fed when these amino acids are present at low levels in the medi- um. Accordingly, we assumed that expression of the fused protein is weakly inhibited by the presence of ex- cess Pro or Leu in the broth.

Growth inhibition by Leu In order to investigate whether Pro or Leu inhibited the expression of the fused protein, we cultivated E. coli HBlOl/pLSDl in 21 of a 0.7% yeast extract in a medium containing 750/lg/ml Leu (or Pro), with feeding 50,~g/ml Pro (or Leu) at the time when the DO level increased. Feeding Leu gave simi- lar results as that shown in Fig. 5 (data not shown). However feeding Pro resulted in a large decrease in the cell growth and the level expression of the fused protein (Fig. 6). These data indicate that this inhibition might be caused by the presence of excess Leu in the broth. These results are consistent with those of experiments reported by Mizutani et al. (25) in which the growth of E. coli

100

g 80

0” 60

Q 40

20

lOoC

7

0 2 4 6 8

Time (h)

800

3 600 3

.j

400 g x lz

200

FIG. 6. Cultivation of E. coli HBlOl/pLSDl with successive addition of Pro. The E. co/i was cultivated in a 0.7,‘% yeast extract medium containing 750 pg/ml Leu (2 r) in a 5 / fermentor. The arrows indicate the times when 50 Pg/ml Pro (final concentration) was added. Open circles, OD; closed circles, the fused protein.

132 NOGUCHI ET AL. J. FERMENT. BIOENG.,

TABLE 2. Effects of amino acid addition on growth of E. coli HBlOl/pLSDl

Addition of amino acids OD at 600 nm

Non-addition 0.08 Val(250 mg/n 0.08 Be (250 mg/l) 1.66

Val(250 mg/f) + lle (250 mg/f) 1.84

E. coli HB IOl/pLSDl was cultivated at 37°C for 16 h in a modified minimum medium that consisted of 0.19; Leu, 0.005% Pro, 0.5?0 glucose, 0.0025% ampicillin, 0.7% Na,HPO,- 12H20, 0.79; KHzP04, 0.7% K2HP04, 0.12% (NH&Sod, 0.0206 NHdCl, 0.1136 MgS04.7H20, 0.00118b’ FeS0,.7H,O, O.OOll”d CaC1,_.2H20, 0.003’4 MnS04.nHZ0, 0.000396 A1Clj.6H10, O.OOOlZ~< CoC&.

6H2G, 0.00006% ZnS04. 7Hz0, 0.00006ad NaZMoOd. 2Hz0, 0.00003% CuS04.5H20, 0.000015”~ H,B03 (pH 7.0).

C600 (leuB6) harboring a recombinant plasmid, pBR322- T. leu, was inhibited by the presence excess Leu in a syn- thetic medium. These authors attained high cell density cultivation of the E. coli C600 by controlling the Leu concentration in the broth. However, we investigated an alternative way, feeding amino acids that prevent the growth inhibition caused by Leu.

Effects of Val and Ile We postulated that excess Leu prevents the biosynthesis and/or the metabolism of Leu, Val or Ile. Therefore, E. coli HBlOl/pLSDl was cultivated in a modified minimum medium containing 1 .O g/l Leu and 50 pg/ml Pro (Table 2). Although the E. coli did not grow well in this medium alone, it grew sufficiently well in this medium supplemented with Ile and Val or Ile only. These results are consistent with those of experiments reported by De Felice and Levin- thal (26) and Tavori et al. (27), and indicate that excess Leu suppresses the biosynthesis of Ile in the bacteria. The addition of Ile prevents the inhibition caused by Leu. Accordingly, it seemed to be plausible that the method used in the experiment of Fig. 5 could be sim- plified if additional Ile was present in the cultivation broth. Therefore, we cultivated E. coli HBlOl/pLSDl in a 0.7% yeast extract medium that contained 0.155% each of Leu, Ile and Pro (Fig. 7) in a 1501 fermentor. During fermentation, 0.04% each of Leu, Ile and Pro was added twice (at 7 and 7.5 h) to maintain the amino acid levels. Addition of Ile prevented the growth inhibi- tion induced by Leu and the cell density reached a final OD at 600nm of 50.4. The amount of the fused protein expressed was 1.24 g/l broth.

Further investigation of scale-up revealed that 1.05% yeast extract in the broth was optimal in the case of a 2,000 I fermentor, since a higher DO level could be main- tained in this fermentor than in the 150 I fermentor. The yield of the fused protein reached 3.Og/l in the 1.05% yeast extract medium in the 2,000 I fermentor. This result may indicate that this fermentation method is con- venient and promising for large-scale industrial produc- tion of recombinant protein expressed by E. co/i HBlOl.

ACKNOWLEDGMENTS

We thank Professor M. Niwa (Tokushima University) for his helpful advice. We are also grateful to Dr. M. Yamashita (Osaka University).

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.l! I I ’ I ! I I 0 2 4 6 8 10 12’

Time (h)

FIG. 7. Cultivation of E. co/i HBlOl/pLSDl with addition of lle, Leu and Pro. The E. coli was cultivated in a 0.7?4 yeast extract medium in a 150 I fermentor. The cultivation conditions are described in Materials and Methods. Open circles, OD; closed circles, the fused protein.

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