Production of streptavidin in a synthetic medium

Journal of Immunological Methods, 113 (1988) 75-81 Elsevier

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JIM 04886

Production of streptavidin in a synthetic m e d i u m * John Cazin, Jr., M. Suter * * and J.E. Butler Department of Microbiology, University of Iowa, lowa City, IA 52242, U.S.A. (Received 20 July 1987, revised received 12 April 1988, accepted 26 April 1988)

A simple, inexpensive procedure for producing streptavidin has been described. The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing L-asparagine as the sole nitrogen source. With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin. When harvested after 7-8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products. Maximal production of streptavidin (100-120 mg/1) was obtained in 8-10 day cultures. For some applications, the culture fluid can be used directly as a source of streptavidin. Under the same conditions used to grow S. avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein. Key words: Streptavidin; Streptomyces avidinii

Introduction

Streptavidin was first produced and isolated by Stapley et al. (1963), who isolated it from broth culture media in which Streptomyces avidinii had grown. The compound was found to be part of a new synergistic antibiotic complex, antibiotic MSD-235, which could be produced in either, a high-nutrient complex natural medium or in a low-nutrient, almost synthetic medium. Subsequently, Chaiet et al. (1963) in studying the antibiotic complex found it to contain two synergistic components that could be obtained in

Correspondence to: J. Cazin, Jr,, Department of Microbiology, University of Iowa, Iowa City, IA 52242, U.S.A. * Supported in part by USDA Grant CRSR-2-2455 and NIH Grant HL 22676. ** Current address: Department of Microbiology and Immunology, University of Illinois College of Medicine at Chicago, ( M / C 790) Box 6998, Chicago, IL 60680, U.S.A.

crystalline from. One was found to be a compound of small molecular weight (< 400), active in Chemically defined media, and capable of inhibiting biotin synthesis by gram-negative organisms. The other component was a protein of large molecular weight (60 000), and was found to form a biologically inactive complex with free biotin. Because of the similarity of biotin-binding by the large molecular weight protein with that of eggwhite avidin, they named the substance streptavidin (SA). Recently, Argarana et al. (1986), who have cloned the SA gene from S. avidinii, also demonstrated a high degree of structural similarity between SA and chicken egg-white avidin. Applications for the avidin-biotin or SA-biotin complexes in various biological fields are numerous, as noted by Wilcheck and Bayer (1984). A need has arisen for increased production of either substance at a moderate cost in order to make it available to most investigators. Some applications, such as the protein-avidin-biotin capture system

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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(PABC) used by Suter and Butler (1986) require sizeable quantities of the biotin-binding compound. Low yields of SA, such as the 10-15 m g / 4 liters culture medium A obtained by Hofmann et al. (1980), make use of the biotin-binding protein quite cumbersome and time-consuming. We describe a simple method for producing reasonably large quantities of SA that is free of contaminating proteinaceous substances present in conventional culture media.

Materials and methods

Culture Streptomyces avidinii (ATCC no. 27419) was obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, M D 20852. Working stock cultures of the organism were maintained on potato dextrose agar (Difco) slants incubated at room temperature (23 ° C). Storage stock cultures were prepared on potato dextrose agar slants in screwcap tubes, by allowing the cultures to incubate for 2 weeks at room temperature, and overlayering the entire slant with sterile mineral oil. The storage stock cultures were stored at room temperature. Production stock cultures were maintained on the synthetic liquid media described below.

Survey cultures A selection of 11 actinomycetes and 134 eumycetes surveyed for SA production came from a stock culture collection maintained in the Department of Microbiology at the University o f | Iowa.

Synthetic liquid media for storage stock cultures and for streptavidin production Trace element and basic salts mixture. H3BO 3 500/~g; CuSO 4 • 5 H 2 0 40/~g; KI 100/~g; FeC13 • 6 H 2 0 200 /~g; MnSO 4. H2 O 400 /~g; NaMoO42 H 2 0 200/~g; ZnSO 4 • 7 H 2 0 400/tg; K H z P O 4 1.0 g; MgSO 4 • 7 H 2 0 0.5 g; NaC1 0.1 g; CaC12 • 2 H 2 0 0.1 g. All components were combined, triturated with a mortar and pestle, and blended in a Waring blender to insure thorough mixing. The mixture was stored in a desiccator jar over anhydrous CaSO4. This mixture of trace elements and basic

salts (TE & BS) was incorporated into the synthetic liquid media at a concentration of 1.7 g/1. Synthetic liquid medium A (SLMA). L-asparagine (cell culture tested, Sigma Chemical Co., St. Louis, MO 63178) 7.0 g; dextrose 10.0 g; TE & BS 1.7 g; K 2 H P O 4 1.0 g; distilled water 1000 ml. The p H was 6.5 after autoclaving. Synthetic liquid medium B (SLMB). L-asparagine (cell culture tested, Sigma Chemical Co., St. Louis, MO 63178) 7.0 g; dextrose 10.0 g; TE & BS 1.7 g; K z H P O 4 1.0 g; K H 2 P O 4 1.0 g; MgSO 47 H 2 0 0.5 g; NaC1 0.1 g; C a C 1 2 . 2 H 2 0 0.1 g; distilled water 1000 ml. The p H was 6.1 after autoclaving. The synthetic liquid media, in volumes of 100 m l / 5 0 0 ml Erlenmeyer flask, were sterilized by autoclaving at 15 lb pressure/15 rain.

S. avidinii inoculum for synthetic liquid medium cultures An inoculum taken from a working stock culture, was introduced into a flask of Stapley liquid medium A, placed on a gyrorotary shaker (New Brunswick Scientific Co., New Brunswick, NJ 08903) at 200 rev/min, and incubated at room temperature until heavy growth in the form of 'balls' appeared. Growth from this culture was transferred to SLMA and SLMB media, and incubated as shake cultures at room temperature. Production stock cultures were prepared by inoculating fresh flasks of SLMA and SLMB media, at weekly intervals, with 5.0 ml of a cell suspension taken from 1-week-old shake cultures grown in the respective culture media. When preparing a 'run' for optimal SA production, the inoculum was prepared from a 4-day-old production stock culture grown on SLMB. The suspension was centrifuged at 500 x g for 15 min, the supernatant fluid was discarded, and the sediment was resuspended in an equal volume of saline. The inoculum consisted of 0.2 ml packed cells/100 ml SLMA. The inoculated culture flasks were placed on the gyrorotary shaker and incubated at room temperature for 7-8 days.

Inoculum for survey cultures A flask of SLMA was inoculated with a small amount of surface growth taken from each of the survey cultures. The inoculated flasks were placed

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on the gyrorotary shaker and incubated at room temperature for 10-14 days.

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Assay for SA production

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The assay for SA production was carried out utilizing an ELISA system described by Suter and Butler (1986), and modified according to Suter et al. (1987). Briefly, this consisted of using a SAbridge-ELISA and a SA-inhibition-ELISA to check for the ability of SA to crosslink biotinylated peroxidase to biotinylated rabbit gamma globulin adsorbed to plastic, and to check for the ability of SA to bind biotin. During a production run, 5 ml samples of culture fluid were removed from culture flasks at daily intervals. After centrifugation, the clear supernatant fluid was decanted, 1 0 / ~ l / m l of a 100 m M solution of N a N 3 was added, and the specimen was stored at 4 ° C until used in a SA assay. Such specimens were found to retain their biotinbinding activity for at least 4 months.

Extraction and purification of SA from cuhure fluids Shake cultures, 7-8 days of age, were centrifuged at 1500 × g for 20 min to sediment the cell growth. The supernatant fluid was removed by aspiration, and SA was separated from other cell substances by ammonium sulfate precipitation, centrifugation and dialysis according to the method of Suter et al. (1988). Purified SA was preserved either in a lyophilized form or it was stored in 50% glycerol at - 20 o C.

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Fig. 1. Comparison of streptavidin production in Stapley medium A ( o o), Bacto vitamin-free yeast base (Difco) (* . . . . . . *), and L-asparagine-modified vitamin-free yeast base ( e . . . . . . e).

Bacto vitamin-free yeast base (Difco), and (2) a modified Bacto vitamin-free yeast base (Difco) medium in which the inorganic nitrogen source, (NH4)2SO4, was replaced with L-asparagine. SA production in these media, compared with produc-

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Culture conditions for SA production An inoculum from a working stock culture of S. avidinii was transferred to a flask of Stapley's liquid culture medium A (Stapley et al., 1963), placed on a gyrorotary shaker and incubated at room temperature. Good growth of the actinomycete, in the form of 'balls' or 'pellets', occurred within a matter of several days. Assays carried out on culture supernatant fluid revealed that the organism produced the biotin-binding protein SA. An attempt to produce streptavidin in a synthetic culture medium was undertaken by inoculating two different synthetic culture media: (1)

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Fig. 2. The effect of L-asparagine concentration on streptavidin production in synthetic liquid medium A (SLMA): 0.1% ( i . . . . . . A); 0.3% (O O); 0.5% (zx zx); 0.7% ( o . . . . . . o ) ; 0.9% ( I - . . . . . It).

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tion in Stapley's liquid medium A, is shown in Fig. 1. Although S. aoidinii was able to grow, albeit much more slowly, in the Bacto vitamin-free yeast base medium, no detectable SA was produced during an observation period of 7 days. In the modified culture medium, after 7 days, SA production was found to be about ten-fold greater than it was in the Stapley liquid culture medium A. Once it has been determined that the asparagine-modified synthetic medium could be used as

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Fig. 4. The effect of inoculum size on streptavidin production in synthetic liquid medium A (SLMA). Milliliters of packed cells/140 ml culture medium: 0.1 (© ©); 0.25 ( e . . . . . . e); 0.5 (/, zx); 1.0 (, . . . . . . ,L); 2.0 (B B).

the production medium, several culture parameters were examined to determine optimal conditions for SA production. Fig. 2 shows that a final concentration of 0.7% L-asparagine proved to be optimal as a nitrogen source for the organism.

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TABLE I ACTINOMYCETES PRODUCTION

ASSAYED

Nocardia asteroides Nocardia barsiliensis Nocardia farcinica Nocardia opaca Nocardia pellegrino Nocardia rubra Nocardia rubra Nocardia sp. Nocardia sp. Streptomyces avidinii Streptomyces griseus Streptomyces rubrireticuli

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Carbohydrate content of the culture medium was found to appreciably influence SA production (Fig. 3), as did the size of initial inoculum used in a production culture (Fig. 4) and the amount of TABLE II EUMYCETES (MOLDS) ASSAYED FOR STREPTAVIDIN PRODUCTION Absidia corymbifera A Ilescheria boydii Allescheria boydii A lternaria alternateria Arthroderma benhamiae (a) Arthroderma benhamiae (A) Arthroderma simii Arthroderma oanbreuseghemii Aspergillus fumigatus Aspergillus niger Aspergillus sp. Aspergillus toxicarius Cephaliophora sp. Cephalosporium sp. Ceratocystis minor Chaetomium reflexum Chrysosporium asperatum Chrysosporium pannosum Circinella sp. Cladosporium carrionii Cladosporium sp. Cunninghamella blakesleeana ( + ) Cunninghamella blakesleeana ( - ) Curvularia sp. Emmonsia parva Epicoccum nigrans Epidermophyton floccosum Epidermophyton floccosum Exophiala werneckii Fonsecaea compactum Fusarium sp. Fusidium sp. Gliocladium sp. Gymnoascus brevisetosus Gymnoascus bruneus Helicoma sp. Helminthosporium sp. Hormodendrum sp. Memnoniella sp. Microsporum audouini

Microsporum canis Microsporum cookei ( + ) Microsporum cookei ( - ) Microsporum distortum Microsporum ferrugineum Microsporum gallinae Microsporum gypseum Microsporum nanum Microsporum vanbreuseghemii ( + ) Microsporum vanbreuseghemii ( - ) Monosporium apiospermum Mortierella wolfii Mucor hiemalis ( + ) Mucor hiemalis ( - ) Nannizzia persicolor Nigrospora sp. Oospora lactis Penicillium chrysogenum Penicillium notatum Phialophora pedrosoi Phialophora verrucosa Phoma sp. Piedraia hortae Polyporus circinatus Pullularia pullulans Radiomyces spectabilis Rhizopus oligosporus Rhizopus sp. Scopulariopsis sp. Sepedonium sp. Spicaria elegans Spicaria oiolacea Sporothrix schenckii Stachybotrys sp. Syncephalastrum sp. Syncephalastrum racemosu~ Trichoderma viride Trichophyton ajelloi Trichophyton concentricum Trichophyton equinum Trichophyton gourvilii Trichophyton megnini

(Table II continued) Trichophyton mentagrophytes Trichphyton rubrum Trichophyton schoenleini Trichophyton soudanense Trichophyton terrestre Trichophyton tonsurans

Trichophyton oerrucosum Trichophyton violaceum Trichothecium roseum Verticillium terrestre Wangiella dermatitidis

Zygorhynchus sp.

aeration that the culture received during growth (Fig. 5). No difference in the amount of growth, or in the amount of SA produced was observed in a medium lacking the three amino acids (L-histidine, DL-methionine and DL-tryptophan) normally included in Bacto vitamin-free yeast base medium. Moreover, growth of S. avidinii and production of SA by the organism was not influenced by addition of either: (a) a combination of threonine, phenylalanine and arginine reported by Chaiet and Wolf (1964) to be major constituents of SA, or by (b) a combination of all other amino acids present in SA. The findings led to the final formulation of SLMA and SLMB culture media, and allowed us to achieve routine production levels in the range of 100-120 mg/1 streptavidin. The quantity of SA produced in either of the two media was essentially the same. Recovery of SA from the SLMB medium yielded only about 2/3 of the SA present in culture supernatant fluid; whereas, when SLMA culture medium was used, greater than 95% recovery was achieved. Stock culture maintenance

Storage stock cultures of S. avidinii, maintained on potato dextrose agar slants covered with sterile mineral oil, were found to be viable and typical in appearance after 16 months of storage at room temperature. Throughout the same period of time, our production stock cultures were maintained in liquid culture on SLMB. Production stock cultures freshly prepared from working stock cultures, maintained on potato dextrose agar slants, were compared with production stock cultures that had been continuously maintained in SLMB for 6 months. No difference was observed in the quan-

80 TABLE III EUMYCETES (YEASTS) ASSAYED FOR STREPTAVIDIN PRODUCTION

Candida albicans Candida quilliermondi Candida krusei Candida lipolytica Candida parapsilosis Candida pseudotropicalis Candida stellatoidea Candida tropicalis Candida zeylanoides Cryptococcus albidus Cryptococcus diffluens Cryptococcus laurentii Cryptococcus luteolus Cryptococcus neoformans Cryptococcus neoformans Cryptococcus neoformans (Type A) Cryptococcus neoformans (Type B) Cryptococcus neoformans (Type C) Cryptococcus neoformans (Type D) Geotrichum sp.

Hansenula ciferri Hansenula saturnus Lipomyces starkeyii Rhodotorula glutinis Rhodotorula graeilis Rhodotorula longissima Rhodotorula muctlaginosa Saccharomyces cerevisiae Saccharomyces fragilis Saccharomyces sp. Saccharomyces sp. Torulopsis candida Torulopsis glabrata Torulopsis magnoliae Tremella aurantia Tremella encephala Tremella mesenterica Trichosporon capitatum Trichosporon cutaneum

tity of SA produced when the organisms were inoculated into SLMA for a production run.

Survey of microorganisms for biotin-binding substances A total of 11 other actinomycete isolates and 134 eumycete isolates were surveyed for their ability to produce an extracellular biotin-binding compound similar to that produced by S. avidinii. All isolates tested lacked the capacity to produce detectable amounts of such a protein. A listing of the organisms tested for SA production is shown in Tables I-III.

Discussion Although S. avidinii was found to grow well in the semisynthetic medium of Stapley et al. (1963), SA yields were consistently low. In order to readily achieve isolation of SA in quantity, large volumes of culture fluid and a specialized purification technique such as the high-capacity immunobiotin column described by Bayer et al. (1986) must be used. We have formulated a synthetic culture medium consisting of L-asparagine,

dextrose and inorganic ions that will produce good growth of S. avidinii, and SA production in the range of 100-120 mg/1 in 8 days. A concentration of 0.7% L-asparagine in the culture medium produced a higher yield of SA than did higher or lower concentrations; although, SA was produced over a range of 0.1-0.9% Lasparagine. Likewise, the optimal dextrose content of the medium was found to be 1%. The final pH of the culture medium was arbitrarily selected, and variations of this parameter were not examined. Overall growth was best, and good yields of SA were obtained in SLMB medium, containing larger amounts of inorganic salts; but, recovery of SA from this medium was not as successful as when SLMA medium was used. Generally, about 60-70% of the SA present in SLMB culture supernates could be recovered; whereas, recovery approached 90-95% from SLMA supernates. There was a cell yield advantage in using SLMB cultures to prepare inocula for SLMA medium production runs; and, the inoculum size influenced the rate and quantity of SA produced over the incubation period. An inoculum of 0.25 ml packed cells, grown in SLMB, per 140 ml SLMA produced considerably better yields than did smaller or larger inocula. SA production was further enhanced by providing for more efficient aeration of the culture. In actively growing cultures, streptavidin was still present in culture fluids for at least 17 days after the culture was inoculated. The rate of production usually began to diminish after about 7-8 days of incubation; and, at that time, cellular breakdown products that interfered with purification of SA began to appear in the medium. Chaiet and Wolf (1964) have suggested that SA may be an enzyme necessary for utilization of biotin. If so, one might expect the substance to be produced in small amounts intracellularly and in close proximity to sites where biotin is used in the fixation of carbon dioxide. With the enormous amount of SA being produced extracellularly by S. avidinii, it is very difficult to imagine such a role, particularly in view of the fact that we were not able to detect such a biotin-binding protein being produced by any one of 11 other actinomycete isolates (representing two genera), or by 134

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eumycete isolates (representing 65 genera) that were assayed. It is likely that this particular strain of S. avidinii has a metabolic defect that allows for an excessive output of a metabolite that usually would be produced in very small amounts by normal cells. This study describes a simple, inexpensive procedure for producing SA by growing S. avidinii in a synthetic culture medium. Our objective was to establish a method of producing SA that would minimize or eliminate extraneous proteinaceous contaminants found in culture media containing yeast extract or peptones. By producing SA in a synthetic culture medium, all proteinaceous constituents present in the culture fluid will have been produced by S. avidinii. When cultures of S. avidinii grown in SLMA are harvested after an incubation period of 7-8 days, streptavidin is relatively free of contaminating cell breakdown products; and, the centrifuged culture supernatant fluid can be used directly in an ELISA test without further purification.

References Argarana, C.E., Kuntz, I.D., Birken, S., Axel, R. and Cantor, C.R. (1986) Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res. 14, 1871-1882.

Bayer, E.A., Ben-Hur, H., Gitlin, G. and Wilchek, M. (1986) An improved method for the single-step purification of streptavidin. J. Biochem, Biophys. Methods 13, 103-112. Chalet, L. and Wolf, F.J. (1964) The properties of streptavidin, a biotin-binding protein produced by streptomycetes. Arch. Biochem. Biophys. 106, 1-5. Chalet, L., Miller, T.W., Tausig, F. and Wolf, F.J. (1963) Antibiotic MSD-235. II. Separation and purification of synergistic components. Antimicrob. Agents Chemother. 3, 28-32. Hofmarm, K., Wood, S.W., Brinton, C.C., Montibeller, J.A. and Finn, F.M. (1980) Immunobiotin affinity columns and their application to retrieval of streptavidin. Proc. Natl. Acad. Sci. U.S.A. 77, 4666-4668. Stapley, E.O., Mata, J.M., Miller, I.M., Demny, T.C. and Woodruff, H.B. (1963) Antibiotic MSD-235. I. Production by Streptomyces avidinii and Streptomyces lavendulae. Antimicrob. Agents Chemother. 3, 20-27. Suter, M. and Butler, J.E. (1986) The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies. Immunol. Lett. 13, 313-316. Suter, M., Cazin, Jr., J., Butler, J.E. and Mock, D. (1988) Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium. J. Immunol. Methods 113, 83. Wilchek, M. and Bayer, E.A. (1984) The avidin-biotin complex in immunology. Immunol. Today 5, 39-43.