(Received for publication, July 26, 1995; and in revised form, September 6, 1995)
From the
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 -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.
Streptavidin, a protein produced by Streptomyces
avidinii, binds D-biotin with a remarkably high affinity (K
10
M)(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 -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.
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) .
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.
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). 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
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-C]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 g for 20 min.
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 -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.
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.
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. , Stv-13;
, Stv-25;
, 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-
C]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.
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. , Stv-13;
, Stv-25;
, 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.
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.).