Large-scale purification of recombinant monellin from yeast

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 82, No. 2, 180-182. 1996

Large-Scale Purification

of Recombinant

Monellin from Yeast

IN HO KIM’” AND KOOK JIN LIM? Department

of Chemical Engineering, Chungnam National University, Taejon 305-764’ and Biotechnology Department, LG Chemical Research Institute, Daeduk Science Town 305-335,” Korea Received

25 September

1995/Accepted

11 April

1996

Recombinant sweet-tasting monellin was purified on a large scale (50 grams) from yeast strain ABllO. The purification yield was 45x, and the purity was as high as 95%, as determined by HPLC gel filtration. Since monellin was proved to be a very basic protein (Cagan, R.: Science, 181,32-35, 1973), the basicity was utilized as an efficient purification method. Acid treatment for precipitating yeast proteins made proteins denatured except monellin. [Key words:

recombinant

monellin,

large-scale

purification]

lysate by using the above-mentioned centrifuge. After centrifuging the cell lysate, the supernatant was acidified with 5 M HCl to remove yeast-contaminating proteins. This purification step proved to be very efficient, since most of yeast proteins have isoelectric points around pH 6, while monellin is a very basic protein (pI=9.3) (10). The acid treatment was done twice at pH=4 and pHz3.0. Precipitated yeast proteins were removed with the Cepa centrifuge. Since the supernatant from the previous acid treatment step was insufficiently clear to proceed to the chromatography processes, a microfilter (DC10 hollow fiber system, 0.1 Corn pore; Amicon, USA) was used to clarify the supernatant. The filter had a surface area of 1Osq. ft and the filtration rate of the supernatant was around 40 ml/min. Filtered and clarified solution was further concentrated with a Pellicon system (PLBC 5 sq. ft cassette, MWCO 3,000; Millipore, USA). The concentration step was finished within 4 h and gave the final volume of 2 1. Operating pressures were 20 psig at the inlet and 15 psig at the outlet. A large column (10 cm D x 100 cm L: Spectrum, USA) packed with 8 I Sephadex G-25 was used to change the ionic strength of the buffer solutions. The loading volume was 1 1. In order to perform ion exchange chromatography, 20mM phosphate buffer (pH 7.2) was eluted into the G-25 after loading of the concentrated sample. The eution flow rate was 2.4 I/h, which was fast enough to accomplish buffer change within 3 h. This step was also used to remove salts from the monellin solution obtained after the ion exchange step. Carboxymethyl cellulose gels (1.5 I; Whatman CM52, USA) in a process column (BP 113/ 15, Pharmacia, Sweden) purified monellin to over 95,Oi purity under the following operating conditions-equilibrium and washing buffer: 20 mM, pH 7.2 phosphate buffer; elution buffer: 0.15 M NaCl in equilibrium buffer; cleaning solution: 0.1 M NaOH; sample loading flow rate: 0.8 l/h; loading volume: 5 1. The solution eluted from the CM52 column was desalted with a G-25 column, and concentrated to 200ml with a spiral cartridge (SlY3; Amicon). The concentrated solution was freeze-dried in a pan freeze-drier (Labconco, USA). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was done on loo& acrylamide gels (11). Monellin was detected by Coomasie blue staining. The protein concentration was determined by the Bradford method (12). A

Monellin and thaumatin are two well-characterized sweet proteins (l), respectively were isolated from serendipity and katemfe African berries (2, 3). Both are approximately 100,000 times sweeter than sugar on a molar basis (4). The monellin amino acid sequence has been determined, and its crystal structure of monellin was investigated (5). These studies indicated that monellin consists of two peptides, an A chain of 45 residues and a B chain of 50 residues, held together by weak noncovalent bonds (6). A combined monellin gene has been synthesized encoding the A- and B-chains as a single peptide (7), and utilized to enhance the flavor of fruits and vegetables by expressing chimeric monellin genes in transgenic plants (8). We report here on the large-scale purification of single-chain monellin expressed in a recombinant yeast strain, ABl 10, by using process-scale equipment. Single-chain monellin was synthesized with a DNA synthesizer and put into a yeast vector of the pUC series with GAP or ADH2 promoters by a research team at Lucky Biotech Corporation (7). Among monellin-harboring yeast strains, a strain with the ADH2 promoter (9) was shake-cultured in a leucine-deficient medium and transferred into a 4501 pilot-scale fermentor (Chemap, Switzerland). The shake-culture volume was increased lOO-fold by making two consecutive seed cultures (20 ml to 200m1, and 200ml to 2 r) for 2d. Flasks containing 2 1 leucine-minus broth were the inoculation source for a 20 1 working volume fermentor (New Brunswick, USA) containing YEPD medium (yeast extract 2,Od, peptone lx, dextrose 29:). Twenty liters of 12-hour-old broth was poured into the pilot scale fermentor (working volume: 330 I; medium composition: corn steep liquor 4%, dextrose 3x), and cultured for 15 h to give a final optical density of 13 at 600 nm. The main culture conditions were 1 vvm, 300 rpm, pHz6.4 and 30°C. Yeast broth was pumped at a rate of 110 I/h into a continuous bowl centrifuge (Cepa Z41G, Germany). Harvested cell cake amounted to 7 kg on a wet basis (water content: 80x), and was mixed with 20 1 of distilled water. The diluted cells were passed into a bead mill (Dynomill KD5, Switzerland) with a flow rate of 0.7 I/ min. Disrupted yeast debris was removed from the cell * Corresponding author. On leave at Department of Chemical and Biochemical Engineering, University of California, Irvine, CA 92717, USA. 180

NOTES

VOL. 82, 1996

TABLE Steps

1.

Yields and purities

‘Fo$m (g/0 .-

1. 2. 3. 4. 5. 6. 7.

Cell lysate Sup. of pH4 Sup. of pH3 Microfiltrate G-25 pool CM-52 pool Final G-25

53 15 12.8 4.5 14.9 32.3 7.8

of purification

MEzerflin Volume Monellin (g) (n/lj (0

.-

5.0 5.1 5.2 3.0 11.6 30.7 7.4

24 20 19 20 4.9 1.5 7.3

120 102 99 60 58 55 54

Mol.Wt.

steps Yield (%)

Purity (%)

100 85 82 50 48 46 45

9 33 40 67 79 95 95

densitometer (model 620; Biorad, USA) was employed to determine monellin quantities on SDS gels. HPLC analysis was performed with a gel filtration column (Proteinpak 60; Waters, USA) connected to a HPLC pump (model 6000A; Waters). The elution solution was 0.1 M NaCl in distilled water. Monellin was successfully expressed in transformed yeast cells as glucose was depleted, and ethanol triggered the ADH2 gene. The expression level of monellin was about 10% of the total protein when we estimate the monellin quantity by densitometry. Figure 1 shows SDSPAGE of samples taken from the purification steps. Lane 3 represents the most eminent purification effect with the pH treatment eliminating almost all of the yeast proteins. Purity can be calculated as the ratio of monellin concentration over protein concentration (Table 1). The purity calculated in Table 1 seems to be underestimated because the Bradford method of protein assay more sensitively detects the various peptides of low molecular weights in the earlier stages of purification. The purification yield was 45x, which was a larger value than those of ordinary protein purification processes. This was due to the fact that the purification steps except for the microfiltration membrane process efficiently recovered monellin. The purification yield markedly dropped (from 82,!4 to 50%) in the microfiltration step. During microfiltration, we observed that lipid materials from yeast cells covered the membrane surface, and the proteins seemed to precipitate on to it when they passed through the membrane pores. Membrane cleaning with NaOH solution was found to be critical so as to maintain the filtration rate during each processing. The final purity of 95% was also determined by analytical gel filtration chromatography (Fig. 2). According to the retention volume of 12.2 ml, the molecular weight of monellin was calculated as 10,000 daltons, which is consistent with the reported value (10). This gel filtration method confirmed the final purity determined by the Bradford method and densitometry. As discussed in the previous page, purity determination by protein assay and densitometry was not so exact in the earlier purification steps, but became more exact in the later steps. A purity increase was achieved in the gel filtration as well as in the cation exchange chromatography step. Figure 3 shows that gel filtration chromatography is effective in removing low molecular weight UV-absorbing peptides. The first main peak in Fig. 3 was collected up to 2.5 I, and then pumped into the CM cellulose cation exchange column. A typical CM-52 ion exchange chromatogram was obtained as shown in Fig. 4. The yellow colors originating from yeast are the cause of the UV-absorbing peak in the loading stage. Yellow colors in the loading sample were almost eliminated in the loading and wash-

1

2

3

4

5

181

6

97.4k 66.2~ 45K 31 K

21.5K

14.4K

FIG. 1. Electrophoresis of purification steps. Lane 1: molecular weight marker, lane 3: pH 3 supernatant, lane 4: microfiltrate, lane 5: concentrate by ultrafiltration, lane 6: finally purified monellin.

ing steps. Using the 0.15 M NaCl condition, monellin was eluted as a single peak. Since a step change of ionic strength was used, a tailing phenomenon was observed. The pooled monellin peak was desalted on the G-25 column with distilled water. In this step, the chromatogram showed a single peak. The desalted solution was freeze-dried and weighed to confirm the final monellin quantity in Table 1. The final monellin weight was equal to the quantity determined by densitometry. The final solid sample was white and fluffy in appearance, and tasted sweet.

Retention

time

(min)

HPLC of recombinant monellin produced in yeast. The ^ ._ __~ chromatography was performed on a Protempak 60 (Waters), now rate: 1 ml/min.

182

J. FERMENT.BIOENG.,

KIM AND LIM

A b s

A

1.0

-

0.8

-

0.6

-

Loading

Washing

Elutian

b s 0

2 8

,” 2 b ’ 0 a nm n ‘.

C

c

Y.”

0

5

Volume

(1)

FIG. 3. Gel filtration chromatogram for removing low molecular weight contaminants. The first peak was monellin which was assayed by electrophoresis.

REFERENCES Cagan, R.: Chemostimulatory protein: a new type of taste stimulus. Science, 181, 32-35 (1973). Morris, J. A. and Cagan, R. H.: Purification of monellin, the sweet principle of Dioscoreophyllum cumminsii. Biochim. Biophys. Acta, 261, 114-122 (1972). van der Wel, H. and Loeve, K.: Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus danielli benth. Eur. J. Biochem., 31, 221-225

20

(1)

FIG. 4. Ion exchange chromatogram. The second peak was purified monellin of which purity was determined by eiectrophoresis and densitometer. Loading volume: 5 I; washing volume: 10 I; elution volume: 2 I; loading and washing flow rate: 0.8 I/h; elution flow rate: 1.5 I/h. (1972).

4. Edens,

Plant monellin was similarly purified using salt precipitation and cation exchange chromatography (2). Compared to the plant monellin purification, yeast monellin purification started with more contaminating yeast proteins. These proteins were mainly eliminated in the acid treatment step. The complex mixture of colors originating from the media and yeast cells is considered as a notorious feature hampering the final appearance of fermentation products. We tried purifying monellin without ion exchange chromatography to reduce the purification cost, but the final freeze-dryed monellin was not of good appearance. Cation exchange gels were found to be a good separation medium, differentiating the monellin from the colors. Monellin is so basic that it could strongly adsorb on cation exchange gels, even at neutral pH. Recombinant thaumatin was also purified a Sulfopropyl cation exchange chromatography (13). Thaumatin is another strong basic protein, which could be efficiently purified by the above SP chromatography.

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8.

9.

10.

11.

12.

13.

L. and van der Wel, H.: Microbial synthesis of the sweet tasting plant protein thaumatin. Trends Biotechnol., 3, 61-64 (1985). Ogata, C.. Hatada, M., Tomlinson. G.. Shin. W.-C.. and Kim, S.-H;: Crystai structure of the ‘intensively .sweet piotein monellin. Nature, 328, 739-742 (1987). Bohak, Z. and Li, S.-L.: The structure of monellin and its relation to the sweetness of the protein. Biochim. Biophys. Acta, 427, 153-170 (1976). Kim, S.-H., Kang, C. H., Kim, R., Cho, J. M., Lee, Y. B., and Lee, T.-K.: Redesigning a sweet protein: increased stability and renaturability. Protein Eng., 2, 571-575 (1989). Penarruhia, L., Kim, R., Giovannoni, J., Kim, S.-H., and Fisher, R.: Production of the sweet protein monellin in transgenie plants. Bio/Technol.. 10, 561-564 (1992). George-Nascimento, C., Gyenes, A., Hallo;an, S. M.. Merryweather, J., Valenzuela, P., Steimer, K. S., Masiarz, F. R., and Randolph, A.: Characterization of recombinant human epidermal growth factor produced in yeast. Biochemistry, 27, 797-802 (1988). Morris, J. A., Martension, R., Deihler, G., and Cagan, R. H.: Characterization of monellin, a protein that tastes sweet. J. Biol. Chem., 248, 534-539 (1973). Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685 (1970). Bradford, N. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). Lee, J.-H., Weickmann, J. L., Koduri, R. K., Ghosh-Dastidar, P., Saito, K., Blair, L. C., Date, T., Lai, J. S., Hollenherg, S. M., Kendall, R. L.: Expression of synthetic thaumatin genes in yeast. Biochemistry, 27, 5101-5107 (1988).