Postsecretory Modificationsof Streptavidin
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Biochem. J. (1989) 259, 369-376 (Printed in Great Britain) 369 Postsecretory modifications of streptavidin Edward A. BAYER, Haya BEN-HUR, Yaffa HILLER and Meir WILCHEK* Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel Streptavidin, an extracellular biotin-binding protein from Streptomyces avidinii, exhibits a multiplicity in its electrophoretic mobility pattern which depends both upon the conditions for growth of the bacterium and upon the protocol used in the purification of the protein. The observed structural heterogeneity appears to reflect the action of two types ofpostsecretory molecular events: proteolytic digestion of the intact Mr- 18000 subunit to a minimal molecular size (approx. Mr 14000), and aggregation of the native tetramer into higher- order oligomeric forms. The extent of subunit degradation and/or tetrameric aggregation affects the capacity of a given streptavidin preparation to interact with biotin-conjugated proteins in different assay systems. INTRODUCTION MATERIALS AND METHODS Streptavidin is a non-glycosylated, neutral bacterial Streptavidin preparations protein from Streptomyces avidinii which is remarkably The commercially prepared streptavidin samples used similar in its biotin-binding properties to the positively in this work were kindly provided as gifts by Apcel Ltd. charged egg-white glycoprotein, avidin. The strength of (Slough, Berks., U.K.), Boehringer-Mannheim G.m.b.H. the binding affinity which these two proteins exhibit for (Mannheim, Germany), Calbiochem AG (Lucerne, biotin is the highest recorded between a protein and its Switzerland), Cetus Corporation (Emeryville, CA, ligand (Green, 1975). Despite the apparent lack of U.S.A.), Immuno-search Inc. (Toms River, NJ, U.S.A.), both immunochemical crossreactivity and evolutionary Jackson ImmunoResearch Laboratories Inc. (West relatedness betwen the two proteins, several structural Grove, PA, U.S.A.), Serotec (Kidlington, U.K.), Sigma characteristics are shared. For example, both avidin Chemical Co. (St. Louis, MO, U.S.A.) and Zymed and streptavidin are of similar molecular size and are Laboratories Inc. (San Francisco, CA, U.S.A.). We composed of four subunits, each of which forms received samples of several milligrams from Apcel, Sigma one biotin-binding site. Nevertheless, the amino acid and Boehringer which enabled us to study their respective composition and primary sequences are very different, N- and C-terminal sequences. although a series of short interrupted similar stretches Streptavidin was also prepared in our laboratory by are evident (Argarana et al., 1986). growing 1-10 litre cultures of S. avidinii, and by isolating In addition to the fact that these biotin-binding the protein from the spent growth medium using an proteins offer an intriguing model system for probing the improved iminobiotin-Sepharose affinity column as interaction between a protein and its ligand (Green, described previously (Bayer et al., 1986a). Lot numbers 1975; Gitlin et al., 1987, 1988a,b), the avidin-biotin referred to in the text indicate the product purified from complex provides an extremely useful, versatile system a given fermentation run. Truncated forms of the protein for general application in the biological sciences (Bayer were prepared by subjecting purified samples to extracts & Wilchek, 1980; Wilchek & Bayer, 1984, 1988). Owing of the culture medium or to purified proteases as to its neutral p1 and non-glycosylated structure, strept- described below. avidin has been used as a substitute in such systems, in order to circumvent problems arising from nonspecific binding which sometimes results from the use of egg- Preparation of whole-cell sonic extracts white avidin. S. avidinii was grown in liquid culture (100 ml) at In recent studies, we (Bayer et al., 1986a) and others 30 °C in medium A (Stapley et al., 1963). The cells (Argarana et al., 1986) have demonstrated that the actual were harvested after 24 h and 96 h of growth, and the molecular mass of streptavidin is higher than that respective cell samples were washed three times with reported earlier (Chaiet & Wolf, 1964; Hofmann et al., phosphate-buffered saline, pH 7.4 (PBS), by centri- 1980). It appears that, either during growth or during fugation (15000 g, 15 min). The washed cells were the isolation process, the subunit of streptavidin may resuspended in 15 ml of PBS, homogenized, and have been partially cleaved in some manner. In our sonicated in an ice bath (4 x 1 min at 50 W power; initial work, we also noted a tendency for aggregation of Branson Sonic Power Co., Danbury, CT, U.S.A.). Pro- the streptavidin tetramer. In the present communication, tein concentration was determined (Bradford, 1976), and these two phenomena are further characterized. the sonicated suspension was stored at -20 °C. Abbreviations used: ASM, extract, 50 ",,-saturated-(NH4)2SO4 fraction of cell-free culture fluids; e.l.i.s.a., enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline, pH 7.4; PAGE, polyacrylamide-gel electrophoresis. * To whom correspondence should be addressed. Vol. 259 370 E. A. Bayer and others (NH4)2SO4 fractionation of cell-free culture fluids subunit composition of a given streptavidin sample, the A 5-day cell culture (2 litres) was harvested, and the samples were boiled in sample buffer and subjected to cell-free culture medium was collected. Half (1 litre) was SDS/PAGE on 150 gels. For analysis of the oligomeric dialysed exhaustively and lyophilized. The other half was state of a given streptavidin sample, samples were treated treated with increasing increments of (NH4)2SO4; all in sample buffer at room temperature and run on 100% steps in the following protocol were carried out in ice or gels. at 4 'C. A combination of kits containing molecular mass No precipitate could be detected in solutions con- markers (Sigma) were used as standards: MW-SDS-200 taining up to 40 %0-saturated (NH4)2SO4. At 50 %- and MW-SDS-70L for the oligomer system; MW-SDS- saturated (NH4)2SO4, an ample amount of precipitate 70L and MW-SDS-17 for the monomer system. formed, which, after a 30 min incubation period, was Biotin-binding assays collected by centrifugation, resuspended in a minimal volume of double-distilled water and saved. To the In order to determine the biotin-binding capacity of a supernatant fluids, solid (NH4)2SO4 was added to obtain given streptavidin sample, two different assay systems a 60 %0-saturated solution, and the latter was stored were employed. In one, samples in solution were overnight. The resultant precipitate was collected, and examined by an e.l.i.s.a.-like assay system which exploited the above procedure was repeated successively to obtain the multivalent nature of the streptavidin molecule. In 700- (60 min incubation), 80 o- (overnight incubation) the second approach, the relative biotin-binding capacity and 100 %0-saturated (4 h incubation) (NH4)2SO4 frac- of individual SDS/PAGE-separated bands was deter- tions. The precipitates were each dissolved in distilled mined. water, dialysed exhaustively against distilled water and then once against PBS. Following dialysis, the residual E.l.i.s.a.-like assay. Serial dilutions of streptavidin precipitate in each sample was centrifuged and discarded. samples were incubated in wells of microtitre plates The respective protein concentrations were determined precoated with biotinyl bovine serum albumin. The (Bradford, 1976), and the samples were stored in aliquots biotin-binding activities of the streptavidin samples were at -20 C. then examined by interaction with biotinyl alkaline phosphatase as reported in a previous publication (Bayer Treatment of streptavidin with cell sonic extracts and et al., 1986b). with (NH4)2S04 fractions Biotin-binding of streptavidin components on blots. To 1 mg/ml samples of streptavidin in PBS (0.01 % The relative biotin-binding capacity of streptavidin sodium azide added when required), the desired extract monomers or oligomers was detected on nitrocellulose or fraction was added. Typically, unless otherwise blots following SDS/PAGE separation. The blotted indicated, 200 ,tg of the respective extract was added in bands were treated with biotinyl alkaline phosphatase as 200 ,ul. The interaction was allowed to proceed at room described by Hiller et al. (1987). The signal associated temperature (unless otherwise stated). Aliquots (200 jdl) with streptavidin oligomers usually appeared within were taken at various time periods (e.g., after 1, 3, 7 and into monomeric components 14 days of incubation) and stored at -20 'C pending 30 min; samples separated SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) were usually incubated for 24 h periods after introduction analysis. of the substrate solution. Gel chromatography Treatment of streptavidin with proteases Gel filtration of streptavidin samples (2 mg in 1 ml) All proteases mentioned in the text were purchased was carried out at 4 °C on a Sephacryl S-300 column, from Sigma. To 100 ,ug samples of streptavidin dissolved equilibrated and eluted with 50 mM-Tris/HCl buffer, in 90 ,ul of an appropriate buffer (according to the pH 7.5, at a flow rate of 10 ml/h. The column dimen- manufacturer's suggestion for each protease), a solution sions were 15 mm x 670 mm, and 1.0 ml fractions were (10 #d) containing 2 ,Cg ofthe desired protease was added. collected. The reactions were allowed to take place at 37 'C (except The desired fractions were pooled, dialysed against for pepsin and papain which were incubated at 25 °C). distilled water, and concentrated by lyophilization. The After 15 min and 2 h, aliquots (50 ltl) were removed, samples were stored at -20 °C at a concentration of combined with 25 ,ul of sample buffer, boiled for 10 min 1 mg/ml. and stored at -20 'C pending SDS/PAGE analysis. Sequence analyses SDS/PAGE and blotting The N-terminal sequence of a given streptavidin The conditions for SDS/PAGE and electrophoretic preparation was determined by Edman degradation using transfer on to nitrocellulose filters were described pre- a gas-phase sequencer (three to five cycles per sample).