©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Core Streptavidins
A MINIMUM-SIZED CORE STREPTAVIDIN HAS ENHANCED STRUCTURAL STABILITY AND HIGHER ACCESSIBILITY TO BIOTINYLATED MACROMOLECULES (*)

(Received for publication, July 26, 1995; and in revised form, September 6, 1995)

Takeshi Sano Mark W. Pandori (§) Xiaomin Chen (1) Cassandra L. Smith Charles R. Cantor (¶)

From the Center for Advanced Biotechnology and Departments of Biomedical Engineering and Pharmacology, Boston University, Boston, Massachusetts 02215 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two recombinant core streptavidins were designed and characterized to understand the role of the terminal sequences, present in naturally truncated core streptavidins, in the properties of streptavidin. One recombinant core streptavidin, Stv-25, has an amino acid sequence very similar to natural core streptavidins. The other recombinant molecule, Stv-13, has further truncation of the terminal residues and consists essentially of only the beta-barrel structure characteristic of streptavidin. These recombinant core streptavidins are tetrameric and bind four biotins/molecule, as does natural streptavidin. The solubility characteristics of Stv-13, determined by varying the concentration of ammonium sulfate or ethanol, were almost the same as those of Stv-25 and natural core streptavidin. However, Stv-13 showed an enhanced structural stability compared with Stv-25 and natural core streptavidin. For example, Stv-13 retained greater than 80% of its biotin binding ability after incubation in 6 M guanidine hydrochloride at pH 1.5, under which conditions, Stv-25 and natural core streptavidin retained only about 20% of their biotin binding ability. In addition, Stv-13 showed higher accessibility to biotinylated DNA than natural core streptavidin. Apparently, the terminal regions, present on the surface of natural core streptavidin, spatially hinder biotinylated macromolecules from approaching the biotin binding sites.


INTRODUCTION

Streptavidin, a protein produced by Streptomyces avidinii, binds D-biotin with a remarkably high affinity (K 10M)(1, 2, 3, 4) . This extremely tight biotin binding affinity has made the streptavidin-biotin system a powerful biological tool in a variety of bioanalytical applications(5, 6) . Streptavidin is generally isolated from culture media of S. avidinii. Such streptavidin molecules usually have truncated terminal sequences due to postsecretory cleavage of the terminal regions, which are highly susceptible to proteolysis(4, 7, 8, 9, 10) . Nontruncated or only partially truncated streptavidins tend to form higher order aggregates and thus have poor solubility. In contrast, fully truncated streptavidin, termed natural core streptavidin, is free from aggregate formation and shows high solubility. However, the terminal sequences of natural core streptavidin often differ from preparation to preparation, and this heterogeneity can be seen even within single tetrameric molecules(9) . Thus, many commercial preparations are treated with proteinases to further truncate the terminal sequences and to maximize the homogeneity of the terminal structure.

In addition to numerous biotechnological applications, streptavidin generates considerable protein chemical interest, particularly as an attractive model for studying macromolecule-ligand interactions (11, 12, 13, 14, 15, 16, 17, 18) . The determination of the three-dimensional structure of core streptavidin by x-ray crystallography (19, 20) considerably expanded the understanding of the structural characteristics of this protein at the molecular level. However, no precise information about the structure of the chain termini was obtained in these studies because of the weak densities seen for these regions in the electron density maps. This indicates that the terminal regions, located on the surface of the molecule, are rather disordered and flexible (20) and that the terminal sequences have little contribution to the fundamental properties of streptavidin, such as formation of the stable beta-barrel structure and biotin binding. Apparently, these disordered structures are also responsible for the high proteinase susceptibility of the terminal regions.

The objective of the present work was to produce, by genetic engineering, core streptavidin that has a homogeneous structure. Such structurally homogeneous streptavidin molecules should be very useful in obtaining deeper understanding of the properties and structural characteristics of streptavidin. We were particularly interested in designing a minimum sized core streptavidin, which might have enhanced properties over natural core streptavidins due to the lack of nonfunctional terminal residues. In this work, two recombinant core streptavidins were designed and produced. One recombinant core streptavidin has a structure very similar to natural core streptavidins; the other has further truncation of the terminal sequences, which have no apparent function. These recombinant core streptavidins were characterized to understand the roles of the terminal regions in the properties of streptavidin.


EXPERIMENTAL PROCEDURES

Construction of Expression Vectors

Expression vectors for recombinant core streptavidins were constructed using standard techniques(21) . Oligonucleotide-directed in vitro mutagenesis (22) was used to introduce mutations into the coding sequence for streptavidin.

Two expression vectors, pTSA-13 and pTSA-25 (Fig. 1), were constructed using a cloned natural streptavidin gene (7) as the starting material. pTSA-13 carries a DNA sequence encoding amino acid residues 16-133 of mature streptavidin(7) , while pTSA-25 encodes amino acid residues 14-138. The coding sequences were cloned into pET-3a (23, 24) under the control of the 10 promoter of bacteriophage T7.


Figure 1: Schematic illustration of the structures of various streptavidin constructs. The amino acid sequence is based on Argaraña et al.(7) . Single-letter amino acid codes are used to indicate terminal sequences. A box represents the sequence from Thr-20 to Phe-130. Stv-13 and Stv-25 are recombinant core streptavidins designed in this work. The structure of natural core streptavidin, obtained from Boehringer Mannheim, is from Bayer et al.(10) .



Expression and Purification of Recombinant Core Streptavidins

Expression of each recombinant core streptavidin was carried out by the T7 expression system using BL21(DE3)(pLysE) (24) carrying an expression vector, as described previously(25, 26, 27) .

Purification of Stv-13 and Stv-25 was carried out by the method described previously(25, 26, 27) , including 2-iminobiotin affinity chromatography(28) . BL21(DE3)(pLysE) carrying pTSA-13 or pTSA-25, which had been incubated for 4 h after induction, was used as the source.

Determination of Solubility Characteristics

The solubility characteristics of recombinant core streptavidins, Stv-13 and Stv-25, without or with biotin were determined by varying concentrations of ammonium sulfate or ethanol. Natural core streptavidin (Boehringer Mannheim) was also analyzed for comparison.

For analysis in the absence of biotin, the concentration of each core streptavidin was adjusted to 5.7 nmol of subunit/ml in Tris-buffered saline (150 mM NaCl, 20 mM Tris-Cl (pH 7.4), 0.02% NaN(3)). This corresponds to 72 µg/ml for Stv-13, and 76 µg/ml for Stv-25 and natural core streptavidin. To 100 µl of this protein solution, 1.1 ml of an appropriate ammonium sulfate solution in Tris-buffered saline was added to adjust the final concentration of ammonium sulfate (final streptavidin concentration, 0.48 nmol of subunit/ml). The mixture was allowed to stand at 30 °C for 30 min and centrifuged at 2,200 times g for 20 min. The amount of soluble streptavidin in the supernatant fraction was determined by biotin binding assays described below. The fraction of original streptavidin remaining in the supernatant is defined as the relative solubility.

For analysis in the presence of biotin, the procedure was almost the same as above, but the biotin binding sites of each core streptavidin were saturated by adding an equimolar amount of D-[carbonyl-^14C]biotin (53 mCi/mmol; Amersham Corp.) prior to the addition of an ammonium sulfate solution. The amount of soluble streptavidin in the final supernatant was estimated from the radioactivity derived from bound biotin, determined by liquid scintillation counting.

When ethanol was used, the procedures were essentially the same as those used with ammonium sulfate, but the following modifications were made. After the addition of ethanol, the final volume was adjusted to 1.2 ml by the addition of an appropriate ethanol solution to make the final protein concentration constant for all samples. After incubation at 30 °C for 30 min, centrifugation was performed at 13,000 times g for 20 min.

Stability against Denaturation by GdnHCl^1

The structural stability of core streptavidins was estimated from the biotin binding ability after incubation in GdnHCl solutions at pH 7.4 or pH 1.5. Each of Stv-13, Stv-25, and natural core streptavidin was incubated at 22 °C for 10 min in 500 µl of an appropriate GdnHCl solution (final GdnHCl concentration, 0-6.0 M) at a protein concentration of 270 pmol subunits/ml (1.7 µg/ml for Stv-13 and 1.8 µg/ml for Stv-25 and natural core streptavidin). Then, 1.4 µl (680 pmol) of D-[carbonyl-^14C]biotin was added to each solution. The mixture was incubated at 22 °C for 10 min, and streptavidin-biotin complexes were separated from free unbound biotin using PD-10 columns (Pharmacia Biotech Inc.), which had been equilibrated with the same GdnHCl solution. The amount of radioactive biotin remaining bound to streptavidin was determined by liquid scintillation counting.

Binding Ability for Biotinylated DNA

The binding ability of two core streptavidin species, Stv-13 and natural core streptavidin, for biotinylated DNA was determined by using a 3179-base pair linear double-stranded DNA target in which one of the 3` termini contains biotin. This target DNA was prepared by using an AccI-HindIII fragment of the plasmid pGEM-3Zf(+) (Promega). Biotin was incorporated into the HindIII terminus by fill-in reactions in the presence of a biotinylated deoxynucleotide analog, biotin-14-dATP (Life Technologies, Inc.), as described earlier (29) . Core streptavidin and the biotinylated target DNA were mixed at various ratios in 10 mM Tris-Cl (pH 8.0), 0.1 mM EDTA, and the mixtures were incubated at 37 °C for 90 min followed by electrophoretic separation on 1% agarose gels. DNA was stained with ethidium bromide.

Other Methods

Gel filtration chromatography was carried out at room temperature (22 °C) using a Sephacryl S-300 HR column (1.6 times 85 cm; Pharmacia), as described previously(26, 30) . Biotin binding ability was determined by gel filtration (31) using D-[carbonyl-^14C]biotin and PD-10 columns. SDS-PAGE (32) was carried out using 15% polyacrylamide gels. Proteins were stained with Coomassie Brilliant Blue R-250. The concentration of each streptavidin preparation was determined from the absorbance at 280 nm using the following extinction coefficients (E): Stv-13, 3.55; Stv-25, 3.35; natural core streptavidin, 3.35(33) .


RESULTS AND DISCUSSION

Design of Recombinant Core Streptavidins

Although mature streptavidin has 159 amino acids/subunit(7) , such full-length, nontruncated molecules can rarely be seen under the conditions generally used for the culture of S. avidinii. This is due to very high susceptibility of the terminal regions of the full-length molecule to proteolysis. Such full-length and only partially truncated molecules tend to form higher-order aggregates and have poor solubility (4, 7, 8, 9, 10) , although the biological reason why S. avidinii produces streptavidin with poor solubility and a tendency to aggregate is unknown. For these reasons, such streptavidins are not useful in bioanalytical applications. In contrast, fully truncated core streptavidins have high solubility and show little tendency to aggregate(4, 7, 8, 9, 10) . Thus, many commercial streptavidin preparations include proteinase treatment to ensure full truncation of the terminal sequences. However, the variable performance seen for natural core streptavidin preparations may be attributable to incomplete truncation of the terminal sequences and residual proteolytic activity, resulting from proteinase treatment(34) .

The objective of the present work was to design recombinant core streptavidin with a homogeneous structure. A particular motivation was derived from the fact that the structural heterogeneity of natural core streptavidins reduces the resolution obtainable in x-ray diffraction studies on streptavidin crystals(19, 20) . We were particularly interested in designing a minimum sized core streptavidin, which might have enhanced properties because of the removal of any nonfunctional terminal sequences that are located on the surface of the molecules.

In this work, two recombinant core streptavidins were designed (Fig. 1). One recombinant core streptavidin, Stv-25, has amino acid residues from Glu-14 to Ala-138 plus a methionine residue at the N terminus, derived from a translation initiation codon, and thus has a structure very similar to natural core streptavidins. Other groups have also produced recombinant core streptavidins(34, 35, 36) , which are very similar to Stv-25.

The other recombinant core streptavidin, Stv-13, has further truncation of the terminal sequences and consists of amino acid residues from Gly-16 to Val-133 plus a methionine residue at the N terminus. Previous crystallographic studies on streptavidin using natural core streptavidin were able to refine the molecular structure only from Ala-13 or Glu-14 to Val-133(19, 20) , which corresponds almost perfectly to the stable beta-barrel structure consisting of the sequence from Gly-19 to Val-133. This implies that the terminal regions of natural core streptavidins have little contribution to the fundamental properties of streptavidin, which should not be altered by the further truncation of the terminal sequences made on Stv-13.

Expression and Purification of Recombinant Core Streptavidins

Expression of Stv-13 and Stv-25 was carried out using the T7 expression system, which allows efficient expression of various recombinant streptavidin constructs(25, 26, 27, 33, 34, 35, 36, 37, 38) . Stv-13 was expressed very efficiently in Escherichia coli as were other recombinant streptavidin constructs(25, 26, 27, 37) . In contrast, the expression efficiency of Stv-25 was considerably lower. This is probably caused by codons for the terminal sequences present in Stv-25 (but absent in Stv-13) that occur at low frequencies in highly expressed E. coli genes. A similar observation was reported with another recombinant core streptavidin(35) , where the expression efficiency in E. coli was rather low with the natural streptavidin gene but significantly improved by using a synthetic gene containing codons observed in highly expressed E. coli genes.

Expressed Stv-13 and Stv-25 were purified to homogeneity (Fig. 2) using a simple procedure that includes 2-iminobiotin affinity chromatography. SDS-PAGE analysis of purified proteins shows a clear difference in subunit molecular mass (650 Da) between Stv-13 and Stv-25. Natural core streptavidin obtained from Boehringer Mannheim also showed a single band on SDS-PAGE, and its migration was very similar to that of Stv-25. Although no terminal sequences were determined on the particular batch, this natural core streptavidin is likely to consist of amino acid residues 13-139, as shown by the terminal sequence analysis of the protein obtained from the same source(10) , because the identity of the terminal residues is determined primarily by the proteinase treatment used.


Figure 2: SDS-PAGE analysis of purified core streptavidins. Lane a, Stv-13; lane b, Stv-25; lane c, natural core streptavidin; lane d, molecular mass standard proteins (Pharmacia). Approximately 2 µg of protein was applied to each lane of a 15% polyacrylamide gel. Proteins were stained with Coomassie Brilliant Blue.



Each of Stv-13, Stv-25, and natural core streptavidin bound greater than 0.96 molecules of biotin per subunit, indicating that these molecules have full biotin binding ability. Gel filtration chromatography using Sephacryl S-300HR showed that each of these core streptavidins is tetrameric and free from aggregate formation.

Solubility Characteristics of Core Streptavidins

Significant differences are observed in solubility characteristics of core streptavidin and full-length or only partially truncated streptavidin(4, 7, 8, 9, 10) . These differences suggest that the terminal regions primarily determine the solubility characteristics of streptavidin. To understand the effect of terminal sequences remaining in natural core streptavidin on the solubility characteristics, the solubility of each core streptavidin species with and without biotin was investigated by varying the concentration of ammonium sulfate or ethanol.

The relative solubility of the three core streptavidins, Stv-13, Stv-25, and natural core streptavidin, as the concentration of ammonium sulfate was altered, showed biphasic changes (Fig. 3, A and B); the solubility decreased sharply with increasing concentrations of ammonium sulfate up to 50% saturation and then increased with further increases in ammonium sulfate concentration. In the absence of biotin, Stv-13 showed slightly lower solubility than Stv-25 and natural core streptavidin at ammonium sulfate concentrations up to 50% saturation, but Stv-13 had the highest solubility at 90% saturation of ammonium sulfate. Biotin binding slightly increased the solubility of the core streptavidins. Similar to the solubility changes without biotin, Stv-13 showed slightly lower solubility than Stv-25 and natural core streptavidin at ammonium sulfate concentrations up to 50% saturation but had the highest solubility at 70 and 90% saturation in the presence of biotin.


Figure 3: Solubility characteristics of core streptavidins in the absence and the presence of biotin. A, ammonium sulfate without biotin; B, ammonium sulfate with biotin; C, ethanol without biotin; D, ethanol with biotin. bullet, Stv-13; circle, Stv-25; up triangle, natural core streptavidin. In the absence of biotin, each core streptavidin solution (100 µl, 5.7 nmol subunits/ml) was mixed with 1.1 ml of an appropriate ammonium sulfate or ethanol solution in Tris-buffered saline. The mixture was allowed to stand at 30 °C for 30 min and centrifuged to remove insoluble materials. The amount of soluble streptavidin in the supernatant fraction was estimated from the biotin binding ability determined by gel filtration(31) . In the presence of biotin, the procedure was almost the same as above, but the biotin binding sites of each core streptavidin were saturated by adding an equimolar amount of D-[carbonyl-^14C]biotin prior to the addition of ammonium sulfate or ethanol. The amount of streptavidin in the final supernatant was estimated from the radioactivity derived from bound biotin. The fraction of original streptavidin remaining in the supernatant to the total is defined as the relative solubility, indicated in percent.



The three core streptavidins showed high solubility (greater than 75%) at ethanol concentrations up to 70% in the absence of biotin (Fig. 3C). At an ethanol concentration of 90%, only about 30% of molecules remained soluble for all of the three core streptavidins. Biotin binding had a slight effect on the solubility (Fig. 3D). There is no marked difference in the solubility characteristics in ethanol among the three core streptavidin species.

Although Stv-13 lacks two charged amino acid residues, Glu-14 and Lys-134, and two polar residues, Ser-136 and Ser-139, there is no significant difference in solubility characteristics among the three core streptavidin species. Although the relative solubilities, determined for each core streptavidin construct, may not indicate the true solubilities because the protein solutions may not have reached equilibrium under the incubation conditions used, these results clearly indicate that the terminal regions of core streptavidins have minimal effects on the solubility characteristics, unlike those of full-length streptavidin.

Structural Stability of Core Streptavidins

One characteristic that has made streptavidin one of the most frequently used proteins in various biological analyses is its extremely high structural stability. This allows, for example, conjugation of streptavidin to partner molecules by using covalent chemistry without disturbing its biotin binding ability. Very tight subunit association of streptavidin also contributes to the overall stability. For example, streptavidin remains tetrameric even in the presence of SDS or urea(33, 39, 40) . The subunit association becomes even tighter upon biotin binding, and the tetrameric structure can be partly maintained by heat treatment in the presence of SDS, under which conditions, streptavidin without biotin dissociates completely into subunits(33) . This tighter subunit association upon biotin binding is essential for maintenance of bound biotin, because dissociated subunits have a much reduced biotin binding affinity due to the lack of intersubunit contacts made by Trp-120 to biotin through the dimer-dimer interface(36, 37) . Extremely harsh conditions are required to effectively release bound biotin from streptavidin. Although the known three-dimensional structure of core streptavidin shows no apparent contact of the terminal residues to biotin, the disordered structure of the terminal regions (19, 20) may affect the overall stability of the protein.

To investigate how the terminal regions affect the overall stability of streptavidin, urea gradient-PAGE (41) was performed using polyacrylamide gels with a urea concentration gradient from 0 to 10 M, along with an acrylamide concentration gradient from 12 to 8%. As a control(41) , urea gradient-PAGE analysis showed a marked decrease in migration of bovine serum albumin at high urea concentrations (data not shown), indicating the unfolding of the molecule. However, the three core streptavidins, Stv-13, Stv-25, and natural core streptavidin, showed no appreciable changes in migration at urea concentrations up to 10 M, indicating the extremely high structural stability of streptavidin. This suggested that more stringent denaturation conditions are needed to allow a comparison of the stability of core streptavidins.

Thus, GdnHCl, a denaturant more potent than urea, was used. At high concentrations and very acidic pH, GdnHCl effectively denature streptavidin and release bound biotin. Biotin-binding ability was used as an estimate of the structural stability. Briefly, each core streptavidin species was incubated in solutions containing various concentrations of GdnHCl at pH 7.4 or 1.5, and then the remaining biotin binding ability was determined by gel filtration (31) (Fig. 4, A and B).


Figure 4: Stability of core streptavidins against denaturation by GdnHCl. bullet, Stv-13; circle, Stv-25; up triangle, natural core streptavidin. Stv-13, Stv-25, and natural core streptavidin (270 pmol of subunit/ml) were incubated at 22 °C for 10 min in GdnHCl solutions at pH 7.4 (A) or 1.5 (B). Then, the biotin binding ability of each core streptavidin was determined by gel filtration(31) . All three core streptavidins bound greater than 0.96 molecules of biotin/subunit at pH 7.4 without GdnHCl, and the biotin binding ability remaining in GdnHCl is indicated in percent.



At pH 7.4, almost no changes in biotin binding ability were observed for all of the core streptavidins at GdnHCl concentrations up to 4 M (Fig. 4A). At 6 M GdnHCl, the biotin binding ability of Stv-25 and natural core streptavidin decreased by approximately 20%, while Stv-13 showed almost no reduction in biotin binding ability, suggesting that Stv-13 has a higher stability against denaturation by GdnHCl than Stv-25 and natural core streptavidin.

The enhanced structural stability of Stv-13 over Stv-25 and natural core streptavidin was observed even more clearly at pH 1.5 (Fig. 4B). Stv-13 retained almost full biotin binding ability at GdnHCl concentrations up to 4 M. In contrast, Stv-25 and natural core streptavidin lost approximately 15% of the biotin binding ability at 4 M GdnHCl. At 6 M GdnHCl, Stv-13 retained greater than 80% of the biotin binding ability, while only about 20% of the biotin binding ability was retained with both Stv-25 and natural core streptavidin.

These results demonstrate that Stv-13 has an enhanced stability against denaturation by GdnHCl when compared with Stv-25 and natural core streptavidin. This implies that the terminal regions reduce the overall structural stability of streptavidin.

Ability of Core Streptavidins to Bind Biotinylated Macromolecules

Full-length or only partially truncated streptavidin has a lower accessibility to biotinylated macromolecules than natural core streptavidins(10) , because of steric hindrance caused by the terminal regions located on the surface of the molecule. To estimate how the terminal sequences of core streptavidin affect the binding to biotinylated macromolecules, the biotinylated DNA-binding ability of two core streptavidin species, Stv-13 and natural core streptavidin, was investigated. Briefly, an end-biotinylated double-stranded DNA target was mixed with core streptavidins at various ratios, and the mixtures were separated by agarose gel electrophoresis followed by staining the DNA targets with ethidium bromide.

Gel electrophoretic analysis of core streptavidin-biotinylated DNA mixtures (Fig. 5) shows that larger amounts of dimeric and trimeric biotinylated DNA targets, which are connected via single streptavidin molecules, were formed with Stv-13 than with natural core streptavidin at any molar ratio of streptavidin subunit to biotin used. Correspondingly, smaller amounts of DNA targets without streptavidin or with single streptavidin molecules (only slightly retarded from free DNA targets that were not well resolved under the electrophoresis conditions used) were observed with Stv-13. Although this analysis is not quantitative, the result indicates that Stv-13 has an enhanced binding ability for biotinylated DNA over natural core streptavidin. The enhanced binding ability of Stv-13 for biotinylated DNA reveals that the terminal regions, present on the surface of natural core streptavidin, sterically hinder the biotin binding sites and prevent biotinylated macromolecules from approaching the biotin binding sites due, presumably, to their disordered structure.


Figure 5: Ability of core streptavidins to bind biotinylated DNA. A 3179-base pair end-biotinylated double-stranded DNA target was mixed with Stv-13 or natural core streptavidin at various ratios and incubated at 37 °C for 90 min. Then, the mixtures were electrophoresed on a 1.0% agarose gel and the DNA targets were stained with ethidium bromide. The left and right lanes for each molar ratio of streptavidin subunit to biotin from 0.25 to 1.0 are with Stv-13 and natural core streptavidin, respectively. The lanes marked 0 and Ex are with the biotinylated DNA target alone and with an excess amount of natural core streptavidin (molar ratio of streptavidin subunit to biotin 1,000), respectively. Each lane contains 290 ng of the biotinylated DNA target. The lane marked M contains a 1-kilobase pair DNA ladder (Life Technologies, Inc.).




FOOTNOTES

*
This work was supported by Grant CA39782 from the National Cancer Institute and the United States Department of Energy Grant DE-FG02-93ER61656. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Depts. of Pathology and Medicine, University of California at San Diego, La Jolla, CA 92039.

To whom correspondence should be addressed: Center for Advanced Biotechnology, Boston University, 36 Cummington St., Boston, MA 02215. Tel.: 617-353-8500: Fax: 617-353-8501.

(^1)
The abbreviations used are: GdnHCl, guanidine hydrochloride; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Arno Pähler, Wayne A. Hendrickson, Francis K. Athappilly, and Sandor Vajda for valuable insights on the three-dimensional structure of streptavidin. We also thank Eric Lee and Lauren Choi for technical assistance at early stages of the project.


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