PROJECT-WORK

OF
“Microbial physiology and metabolism”
(BTY - 538)

ON THE TOPIC :- Streptomyces avidini

SUBMITTED BY:
SHASHI SHARMA
Roll no. – RP8003B15
M.Sc.- microbiology
Reg. no. – 11006142
TABLE OF CONTENTS

Introducation

Objective

Taxonomy – Streptomyces avdinii

ISOLATION AND CHARACTERIZATION OF A PSYCHROTOLERANT

STREPTOMYCES STRAIN FROM PERMAFROST SOIL

• Morphological, physiological and biochemical characteristics of

strain SB9 and Streptomyces avidinii ISP 5526.

INTRODUCTION about streptavidin produced by S.avidinii

o Streptavidin

o Structure

o Origin of the high affinity

o Uses in biotechnology

o Pretargetted Immunotherapy

o Monovalent and monomeric Streptavidin

o Comparison to avidini

Improvement of production, assay streptavidin and purification of

streptavidin

• Production of streptavidin in a synthetic medium

• Procedure for the purification of streptavidin by hydrophobic
interaction chromatography
 • Expression and Purification of Recombinant Streptavidin-Containing
Chimeric Proteins
Culture of Streptomyces and purification of streptavidin
• Binding of biotin to streptavidin stabilizes intersubunit salt bridges
between Asp61 and His87 at low pH.
• Structural studies of the streptavidin binding loop.
• Structural origins of high-affinity biotin binding to streptavidin.
• Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine
residues are involved in the binding site.
• Studies on the biotin-binding site of streptavidin. Tryptophan residues
involved in the active site.
• Molecular cloning and nucleotide sequence of the streptavidin gene.

Streptavidin- biotin binding systems are finding widening applications In

biotechnology,medicines and in environmental studies

Current Research

References

Introducation
Egg white contains many proteins and glycoproteins with unidue properties with
unique properties. One of the most interesting,which binds tenaciously to biotin,
was isolated in 1963. this glycoprotein , called avidin due to its “avid” binding of
biotin , was suggested to play an important role : making egg white antimicrobial
by “tying up” the biotin needed by many micro-organisms. Avidin, which
functions best under alkaline conditions, has the highest known binding affinity
between a protein and ligand. Several Years later, scientists at Merck & Co. ,Inc
. discovered a similar protein produced by the actinomycete Streptomyces avdini
, which binds biotin at neutral pH and which doesnot contain carbohydrates.

Objective

These characteristics make streptavidin produced by Streptomyces avdini, has

an ideal binding agent for biotin, and it has been used in an almost unlimited
range of applications in biotechnology,medicines and in environmental studies
that’s why I have choosen this bacteria for my end term project work.

Taxonomy

superkingdom Bacteria
phylum Actinobacteria
order Actinomycetales
family Streptomycetaceae
genus Streptomyces

ISOLATION AND CHARACTERIZATION OF A PSYCHROTOLERANT

STREPTOMYCES STRAIN FROM PERMAFROST SOIL

Phenotypic and physiological characteristics

Strain SB9 was observed to grow well on Gauze I medium and two ISP agar
media (ISP 4 (salt-starch agar) and ISP 5(glycerol-asparagine agar). No growth
was detected on ISP 2 (yeast-malt agar) and ISP 3 (oatmeal agar). Aerial
mycelium was abundant on Gauze I. It was white to pink in color and the
substrate mycelium was light yellow. No aerial mycelium was observed on ISP 4
where the substrate mycelium was beige. Yellow diffusible pigment was
generated only on Gauze I medium. No melanin production was detected on any
of the tested media. Strain SB9 grows well at temperatures between 4 and 30oC,
but the optimal growth appears between 20 and 28oC. Strain SB9 tolerates NaCl
concentrations up to 7% and grows well in TSB medium adjusted to pH 6.5-8.0.
The strain is capable of utilizing several carbon sources, including arabinose,
cellobiose, fructose, D-galactose, D-glucose, innulin, D-lactose, trehalose,
mannitol, D-mannose, D-melibiose, raffinose, L-rhamnose, salicin, sucrose and
xylose.
Morphological, physiological and biochemical characteristics of strain SB9
and Streptomyces avidinii ISP 5526.

Table 1.
Characteristic SB9 Streptomyces avidinii ISP
5526
Colour of aerial mycelium on
Gauze I grayish pink ND
ISP medium 2 N one Grayish
yellowish pink to
ISP medium 3 N one light
grayish reddish
ISP medium 4 N one brown on
ISP 2, 3, 4, 5
ISP medium 5 beige

Colour of substrate mycelium on:

Gauze I pale yellow
ND
ISP medium 2 N one C olorless or
pale yellow
ISP medium 3 N one to light
yellowish brown
ISP medium 4 white on ISP
2, 3, 4, 5
ISP medium 5 beige

Colour of soluble pigment on:

Gauze I yellow N
D
ISP medium 2 N one N one
ISP medium 3 N one N one
ISP medium 4 N one N one
ISP medium 5 N one
N one

Melanin production on tyrosin agar N one

None
Maximum NaCl tolerance (%, w/v) 7 ND
Growth at: *
4°C + ND
28°C ++ ++

Growth on sole carbon sources

(1%, w/v):
Adonitol - ND
Arabinose - -
Cellobiose +/- ND
Fructose + +
D-galactose +/- ND
D-glucose + +
Innulin - ND
D-lactose - ND
Trehalose +/- ND
Mannitol +/- -
D-mannose - ND
D-melibiose +/- ND
Raffinose +/- -
L-rhamnose - +/-
Salicin +/- ND
Sucrose - +/-
Xylose - -

• -, no growth; +/-, poor growth; +, moderate growth; ++, abundant growth;

ND - not define
Strain SB9 shows strong antibacterial activity against Gram-positive, Gram-
negative bacteria and some fungi. The morphological, physiological and
biochemical characteristics of strain SB9 are shown on Table 1.

Phylogenetic analysis
Amplification reactions of the genomic DNA of strain SB9 with ACT primer set
(243F and A3R) yielded a strong PCR product of expected size 1.25 kb (Fig. 1).
This fragment includes all the positions where the genus- and family-specific
primers are located (16). To confirm that strain SB9 was a streptomycete, we
sequenced the almost-complete 16S rRNA gene (EU878377) and compared it
with the 16S rRNA gene sequences of previously described streptomycetes. A
BLAST search of the partial 16S rRNA sequence (1176 bp) showed 99% of
nucleotide sequence similarity to strain Streptomyces avidinii. This value
corresponds to 8 nt differences out of 1160 positions.

Fig. 1. PCR amplification of a partial 16S rRNA region (1250 bp) of strain SB9
with primer combination 243F/A3R. Lane 1: GeneRuler - 1 kb DNA Ladder
(Fermentas); Lane 2: SB9; Lane 3: positive PCR control (Streptomyces
rimosus)

INTRODUCTION - Streptavidin
Streptavidin is a 52,800 dalton tetrameric protein purified from
the bacterium Streptomyces avidinii. It has an extraordinarily high affinity
for biotin (also known as vitamin B7); the dissociation constant (Kd) of the biotin-
streptavidin complex is on the order of
strongest non-covalent interactions known
molecular biology and bionanotechnology
biotin-binding is resistant to extremes of
denaturants (e.g. guanidinium chloride),
proteolytic enzymes.
Structure
~10-14 mol/L, making it one of the
in nature. It is used extensively in
as, in addition to the high affinity,
pH, temperature, organic solvents,
detergents (e.g. SDS, Triton) and

Monomeric streptavidin (ribbon diagram) with bound biotin (spheres)

Tetrameric structure of streptavidin with 2 bound biotins

The crystal structure of streptavidin with biotin bound was first solved in 1989 by
Hendrickson et al and as of May 2009, there are 134 structures deposited in
the Protein Data Bank. The N and C termini of the 159 residue full-length protein
are processed to give a shorter ‘core’ streptavidin, usually composed of residues
13 - 139; removal of the N and C termini is necessary for the high biotin-binding
affinity. The secondary structure of a streptavidin monomer is composed of eight
antiparallel β-strands, which fold to give an antiparallel beta barrel tertiary
structure. Abiotin binding-site is located at one end of each β-barrel. Four
identical streptavidin monomers (i.e. four identical β-barrels) associate to give
streptavidin’s tetrameric quaternary structure. The biotin binding-site in each
barrel consists of residues from the interior of the barrel, together with a
conserved Trp120 from neighbouring subunit. In this way, each subunit
contributes to the binding site on the neighbouring subunit, and so the tetramer
can also be considered a dimer of functional dimers.
Origin of the high affinity
The numerous crystal structures of the streptavidin-biotin complex have shed
light on the origins of the remarkable affinity. Firstly, there is high shape
complementarity between the binding pocket and biotin. Secondly, there is an
extensive network of hydrogen bonds formed to biotin when in the binding site.
There are eight hydrogen bonds directly made to residues in the binding site (the
so called 'first shell' of hydrogen bonding), involving residues Asn23, Tyr43,
Ser27, Ser45, Asn49, Ser88, Thr90 and Asp128. There is also a 'second shell' of
hydrogen bonding involving residues that interact with the first shell residues.
However, the streptavidin-biotin affinity exceeds that which would be predicted
from the hydrogen bonding interactions alone, alluding to another mechanism
contributing to the high affinity. The biotin-binding pocket is hydrophobic, and
there are numerous van der Waals contacts and hydrophobic interactions made
to the biotin when in the pocket, which is also thought to account for the high
affinity. In particular, the pocket is lined with conserved tryptophan residues.
Lastly, biotin binding is accompanied by the stabilisation of a flexible loop
connecting B strands 3 and 4 (L3/4), which closes over the bound biotin, acting
like a 'lid' over the binding pocket and contributing to the extremely slow biotin
dissociation rate.
Most attempts at mutating streptavidin result in a lowered biotin-binding affinity,
which is to be expected in such a highly optimised system. However, a recently
engineered mutant of streptavidin, named traptavidin, was found to have more
than ten-fold slower biotin dissociation, in addition to higher thermal and
mechanical stability However, this decreased dissociation rate was also
accompanied by a decreased association rate.
Uses in biotechnology
Among the most common uses are the purification or detection of various
biomolecules. The strong streptavidin-biotin bond can be used to attach various
biomolecules to one another or onto a solid support. Harsh conditions are
needed to break the streptavidin-biotin interaction, which often denatures the
protein of interest being purified. However, it has been shown that a short
incubation in water above 70°C will reversibly break the interaction without
denaturing streptavidin, allowing re-use of the streptavidin solid support. A
further application is the so called Strep-tag, which is an optimized system for the
purification and detection

Improvement of production, assay streptavidin and purification of

streptavidin
The production of streptavidin by Streptomyces avidinii in several different media
was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation
media containing either complex or multiple carbon sources resulted in higher
yields of streptavidin than media with a single carbon source. Streptavidin could
be detected in crude fermentation broths by use of a tritiated biotin binding
assay. This assay appears to give useful estimates of streptavidin production.
Depending upon the medium employed, streptavidin yields ranged from 0.5 rag/1
to 53 mg/1. Production was successfully scaled up to ten liter fermentors.
Streptavidin was purified in a one step process from centrifuged, concentrated
fermentation broths by binding the protein to an iminobiotin column at pH 11
followed by elution at pH 4.0. Recovery percentages varied depending upon the
solubility of the fermentation media ingredients.
Streptavidin, secreted by Streptomyces avidinii, is a nonglycosylated neutral
protein that binds four biotins as tightly as egg white avidin. Biomedical systems
but encounter nonspecific binding of the positively charged, glycosylated egg
white avidin to negatively charged membranes and plastics Because our
diagnostic division required large quantities of streptavidin for developing
detection reagents at a time when limited amounts were commercially available,
we evaluated methods for producing high concentrations in a variety of
fermentation media.researchers find many uses for biotin-avidin Although
several methods have been identified for quantitation of avidins , the
heterogeneity of media composition and the production of dark pigments by
Streptomyces avidinii have rendered a spectrophotometric assay difficult without
prior purification of the fermented culture media. Since purification may result in
variable yields, we adapted a radioactive biotin-binding method to measure
streptavidin directly in crude fermentation broths. This method enabled us to
screen a variety of fermentation media for optimal streptavidin production.
simplified method for isolating streptavidin from culture supernatants
MATERIALS AND METHODS
A. Growth and fermentation of Streptomyces avidinii
1. Culture and inoeulum preparation." Streptomyces avidinii ATCC 27419 was
the streptavidin producing strain used in this study . A one cm square agar block
of S. avidinii spores and mycelia, cut from a 10 day slant culture grown on
starchcasein medium at 28~ served as the inoculum for the germination medium
The germination medium (medium F, Table 1) was dispensed in 50 ml aliquots
into 250 ml Erlenmeyer flasks with cotton gauze plug. After 72 h growth at 28~
five percent (v/v) of the germination growth material was transferred to the
fermentation media.
2. Fermentation flask studies: The nine fermentation media tested for optimal
production of streptavidin by S. avidinii Media varied in carbon and nitrogen
sources, and in degree of complexity; i.e. single, multiple or complex carbon
sources. The pH values of the media were between 6.4 and 7.0 after
autoclaving. Fermentation media were dispensed as 50 ml aliquots per 250-ml
Erlenmeyer flasks with cotton gauze plugs. All flasks were incubated at 28~ in a
New Brunswick Psychotherm Rotary Shaker set at 200 rpm, with no humidity
control. Flasks were harvested at 20, 48 and 72 h. Wet weights of cell mass
were determined by weighing the cells after centrifugation at 6000 • g for 20 min.
Cell weight was not deterusing an iminobiotin column determined for medium B,
due to the presence of Ca- CO3.
B. Fermentor study A Chemap ten-liter fermentor was prepared containing nine
liters of medium C without glucose. Filter-sterilized glucose was added to the
sterile medium through a port to a final concentration of 1%. The glucose
concentration was maintained at 1% throughout the run. The fermentor was
inoculatedwith 3.9% v/v of S. avidinii cells grown in germination medium.
Previous flask studies indicated that optimal production of streptavidin occurred
between 28 and 30~ and at pH 6.5, therefore, fermentor conditions were set at
28~ 410 rpm, pH control at 6.5, and air at one liter/rain. Aseptic 20 ml samples
were taken daily for biotin binding assays. The fermentor growth material was
usually harvested at 72 h, depending on ~the biotin binding results. Fermentor
contents were centrifuged and the supernatant was decanted and filtered
through two layers of Whatman # 54 filter paper. The light golden brown
supernatant, containing the desired protein, was either purified immediately or
frozen at - 70~ Two fermentor runs are compared
C. Assay of streptavidin with the tritiated biotin binding assay A sample of
culture medium was centrifuged to remove cells and debris. The supernatant
could be used directly, or diluted into 0.1% bovine serum albumin in phosphate
buffered saline (PBS = 130 mM NaC1, 15 mM KHzPO4, 8 mM NazHPO4, 3 mM
KC1, pH 7.4). The sample was incubated at room temperature for thirty minutes
with an excess of tritiated biotin (New England Nuclear 40 Ci/ retool, labeled in
positions 8 and 9). The total volume of the incubation mixture was 200
microliters: 150 microliters of the mixture was PBS. The mixture was fractionated
on a G-25 Sephadex column (Pharmacia). One milliliter fractions were collected
and 100 microliter samples counted in 10 milliliter PCSII scintillation fluid
(Amersham). the biotin that was bound to large molecules bound biotin
separated reasonably well from the protein bound peak. From the total
radioactivity eluted in the first peak and the specific activity of the tritiated biotin,
the amount of streptavidin could be calculated.
D. Protein purification
1. Concentration: The supernatant from the centrifuged culture was concentrated
tenfold at 4~ reservoir to facilitate processing. Our best results were obtained
with a I0 000 M.W. cutoff filter. The filtrate appeared as a pale yellow fluid and
the concentrate was dark brown. The filtrate had no streptavidin detectable by
the biotin binding assay, whereas the concentrate retained 90% of the
streptavidin assayed in the supernatant.
2. Iminobiotin column." Iminobiotin is a biotin analog (Fig. 3) that binds avidins at
pH 11, but not iminobiotin molecule is uncharged, like biotin, but at low pH the
imine acquires a positive charge which lowers its affinity to avidin by several
orders of magnitude. Pierce (Rockford, IL) provides immobilized iminobiotin in
gel form with a reported capacity of one mg avidin/ml resin. A 2.5 cm diameter by
25 cm column was packed with 90 ml of iminobiotin gel and equilibrated with 50
mM ammonium carbonate at pH 11. The culture concentrate was diluted 1:1 with
100 mM ammonium carbonate and the pH was kept at 11. Approximately 20 mg
of streptavidin was applied to the column, which was washed with pH 11 buffer
until the eluate ceased to absorb at 280 rim. The column buffer was then
switched to 50 mM ammonium acetate, pH 4, to elute the streptavidin. The
streptavidin-containing fractions were pooled, lyophilized, and stored at -20~ Fig.
5 shows a typical spectrum of streptavidin before and after treatment with acetic
acid followed by filtration through a G-25 Sephadex colunto. This acetic acid
treatment enables one to more accurately determine the number of binding
sitesat pH 4 per molecule of biotin since the protein absorbance appears in a
more pure form at 280 nm.
3. Gel electrophoresis: A minigel of 11% acrylamide was polymerized with
ammonium persulfate and prepared with 0.1% SDS Tris HC1, pH 6.8 in the
stacking gel and pH 8.85 in the running gel. Samples of Bethesda Research
Laboratories streptavidin, purified streptavidin produced by S. avidi~ nii as
described, and concentrated fermentation supernatants were boiled in SDS-Tris-
mercaptoethanol before application. The gel was stained with Coomassie blue to
visualize proteins.
RESULTS AND DISCUSSION
A. Flask media studies Media were evaluated on the basis of streptavidin
production, biotin binding sites per molecule and nonspecific residues left on the
iminobiotin affinity column. Of the fermentation media tested, medium H yielded
the highest level of streptavidin per ml mentor runs A and B may be due in part
to a time period between 24 and 36 h when a problem with temperature control
resulted in a temperature decrease. It was decided to continue the fermentation
run in Fermentor
B an additional 24 h to see if the cell,~ could recover and continue to produce
streptavidin. cell recovery was achieved and streptavidin production continued.
The'. amount of streptavidin recovered from Fermentor A after 45 h indicated that
even after two days, reasonably good yields (38.7 rag/l) were produced.
C. Assay The tritiated biotin binding assay as described uncter Materials and
Methods can be used to estimate the concentration of active streptavidin at all
stages of the fermentation and purification. Spikes of streptavidin into culture
media before growth could be quantitated within 10% of the actual value.
Therefore, no materials that significantly interfere with biotin binding are present
in culture media. However, some large molecular weight components of the
media may also bind biotin, but less tightly than streptavidin, and account for the
lower
D. Purification Yields of streptavidin from different media varied, with an
average 40% recovery of biotin binding activity as purified streptavidin. The
elimination of an ammonium sulfate precipitation step, described by Hoffmann,
prevented dark pigments from binding to the iminobiotin column and slowing the
flow rate. The iminobiotin column could be regenerated by extensive washing
with pH 11 buffer. SDS-PAGE gels of the various preparations were run to
confirm purity .Although the major subunit of 18 kDa is diffuse, this characteristic
is also observed in the fully active streptavidin from BRL in lane 2 of Fig. 6. It
may represent proteolytic processing of the subunit and can also be seen in the
crude supernatants. Under these reducing conditions, some dimer of the
streptavidin subunit is still present both in the BRL and our purified streptavidin.
Once the streptavidin had been completley purified, the absorbency coefficient,
Ezs0 (1%) of 34
CONCLUSION
From the results obtained, it is apparent that several media are well suited for the
large scale production of streptavidin. The biotin-binding assay can effectively
monitor streptavidin production, even in very dark media. With the simplified
purification process described, the method may offer a more cost effective
production of streptavid-in. Although the 53 mg/1 yield of streptavidin produced
by S. avidinii under fermentor conditions was approximately half that recently
reported by Cazin, et al. and Suter, et al. our methodology has a two fold
advantage; shorter fermentation time (3 vs. 8-10 days) and no extra purification
steps needed for removal of cellular breakdown products that ciency may be
gained by utilizing the more stable iminobiotin columns described by Bayer et al
Production of streptavidin in a synthetic medium

A simple, inexpensive procedure for producing streptavidin has been described.

The biotin-binding protein was produced by growingStreptomyces 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.

Procedure for the purification of streptavidin by hydrophobic interaction

chromatography

A procedure is described for the purification of hydrophobic microbial proteins

such as streptavidin from Streptomyces avidinii, using Benzyl-DC bead cellulose
as the column material. The separation is rapid with a high loading capacity and
sufficient resolution for preparative uses. Advantages are discussed especially
for industrial purposes.

Expression and Purification of Recombinant Streptavidin-Containing

Chimeric Proteins

Streptavidin, a protein produced by Streptomyces avidinii, binds a water-soluble

vitamin, D-biotin (vitamin H), with remarkably high affinity .The dissociation
constant of the streptavidin-biotin complex is approx 10−15 M; the binding of
streptavidin to biotin is one of the strongest noncovalent interactions found in
biological systems. The extremely tight and specific biotin-binding ability of
streptavidin has made this protein a very powerful biological tool for a variety of
biological and biomedical analyses .The ability of biotin to be incorporated easily
into various biological materials has also expanded the application of the
streptavidin-biotin technology to a wider range of biological systems.

Culture of Streptomyces and purification of streptavidin

For preparation and purification of Streptavidin, two synthetic mediums and a
procedure for purifying Streptavidin were studied. We found that Streptomyces
avidinii grew vigorously and showed typical colony characterization. At the
sametime, these synthetic mediums were in favour of producing Streptavidin with
a maximal production of 15.2 micrograms/ml by using DEAE 52 column for
purification of Streptavidin. This purification procedure is very simple and easy,
and could be widely used.

Binding of biotin to streptavidin stabilizes intersubunit salt bridges

between Asp61 and His87 at low pH.

The remarkable stability of the streptavidin tetramer towards subunit dissociation

becomes even greater upon binding of biotin. At two equivalent extensive
monomer-monomer interfaces, monomers tightly associate into dimers that in
turn associate into the tetramer at a less extensive dimer-dimer interface. To
probe the structural basis for the enhancement of the stability of streptavidin by
biotin, the crystal structures of apostreptavidin and its complexes with biotin and
other small molecule and cyclic peptide ligands were determined and compared
at resolutions as high as 1.36 A over a range of pH values from as low as 1.39.
At low pH dramatic changes occur in the conformation and intersubunit hydrogen
bonds involving the loop comprising Asp61 to Ser69. The hydrogen-bonded salt
bridge between Asp61 Odelta2 and His87 Ndelta1, observed at higher pH, is
replaced with a strong hydrogen bond between Asp61 Odelta1 and Asn85
Odelta1. Through crystallography at multiple pH values, the pH where this
conformational change occurs, and thus the pKa of Asp61, was determined in
crystals of space group I222 and/or I4122 of apostreptavidin and complexes. A
range in pKa values for Asp61 was observed in these structures, the lowest
being 1.78+/-0.19 for I222 streptavidin-biotin in 2.9 M (NH4)2SO4. At low pH the
decrease in pKa of Asp61 and preservation of the intersubunit Asp61 Odelta2-
Ndelta1 His87 hydrogen-bonded salt bridge in streptavidin-biotin versus
apostreptavidin or streptavidin-peptide complexes is associated with an ordering
of the flexible flap comprising residues Ala46 to Glu51, that in turn orders the
Arg84 side-chain of a neighboring loop through resulting hydrogen bonds.
Ordering of Arg84 in close proximity to the strong intersubunit interface appears
to stabilize the conformation associated with the Asp61 Odelta2-Ndelta1 His87
hydrogen-bonded salt bridge. Thus, in addition to the established role of biotin in
tetramer stabilization by direct mediation of intersubunit interactions at the weak
interface through contact with Trp120, biotin may enhance tetramer stability at
the strong interface more indirectly by ordering loop residues.
Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important

biotechnological applications and is an interesting model system for studying
high-affinity protein-ligand interactions. We report here crystallographic studies
elucidating the conformation of the flexible binding loop of streptavidin (residues
45 to 52) in the unbound and bound forms. The crystal structures of unbound
streptavidin have been determined in two monoclinic crystal forms. The binding
loop generally adopts an open conformation in the unbound species. In one
subunit of one crystal form, the flexible loop adopts the closed conformation and
an analysis of packing interactions suggests that protein-protein contacts
stabilize the closed loop conformation. In the other crystal form all loops adopt an
open conformation. Co-crystallization of streptavidin and biotin resulted in two
additional, different crystal forms, with ligand bound in all four binding sites of the
first crystal form and biotin bound in only two subunits in a second. The major
change associated with binding of biotin is the closure of the surface loop
incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical
conformation in unbound subunits of our structures as opposed to the disordered
loops observed in other structure determinations of streptavidin. In addition, the
open conformation is stabilized by a beta-sheet hydrogen bond between
residues 45 and 52, which cannot occur in the closed conformation. The 3(10)
helix is observed in nearly all unbound subunits of both the co-crystallized and
ligand-free structures. An analysis of the temperature factors of the binding loop
regions suggests that the mobility of the closed loops in the complexed
structures is lower than in the open loops of the ligand-free structures. The two
biotin bound subunits in the tetramer found in the MONO-b1 crystal form are
those that contribute Trp 120 across their respective binding pockets, suggesting
a structural link between these binding sites in the tetramer. However, there are
no obvious signatures of binding site communication observed upon ligand
binding, such as quaternary structure changes or shifts in the region of Trp 120.
These studies demonstrate that while crystallographic packing interactions can
stabilize both the open and closed forms of the flexible loop, in their absence the
loop is open in the unbound state and closed in the presence of biotin. If present
in solution, the helical structure in the open loop conformation could moderate
the entropic penalty associated with biotin binding by contributing an order-to-
disorder component to the loop closure.

Structural origins of high-affinity biotin binding to streptavidin.

The high affinity of the noncovalent interaction between biotin and streptavidin
forms the basis for many diagnostic assays that require the formation of an
irreversible and specific linkage between biological macromolecules.
Comparison of the refined crystal structures of apo and a streptavidin:biotin
complex shows that the high affinity results from several factors. These factors
include the formation of multiple hydrogen bonds and van der Waals interactions
between biotin and the protein, together with the ordering of surface polypeptide
loops that bury the biotin in the protein interior. Structural alterations at the biotin
binding site produce quaternary changes in the streptavidin tetramer. These
changes apparently propagate through cooperative deformations in the twisted
beta sheets that link tetramer subunits.

Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine

residues are involved in the binding site.

The involvement of tyrosine in the biotin-binding sites of the egg-white

glycoprotein avidin and the bacterial protein streptavidin was examined by using
the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F).
Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin
subunit caused the complete loss of biotin binding. This indicates that the single
tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-
binding site and that its modification by Nbs also abolishes the binding properties
of a neighbouring subunit. This suggests that the tyrosine residues of the egg-
white protein may also contribute to the stabilization of the native protein
structure. In streptavidin, however, the modification of an average of 3 mol of
tyrosine residue/mol of subunit was required to inactivate completely the biotin-
binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of
subunit was protected in the presence of biotin. The difference between the
h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and
the Nbs-modified streptavidin-biotin complex revealed two additional fractions in
the unprotected protein that contain Nbs-modified tyrosine residues. These
residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute
to the biotin-binding site in streptavidin.

Studies on the biotin-binding site of streptavidin. Tryptophan residues

involved in the active site.

Streptavidin, the non-glycosylated bacterial analogue of the egg-white

glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-
5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding
activity was achieved upon modification of an average of one tryptophan residue
per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified
streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three
major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal
sequence analysis revealed that tryptophan residues 92, 108 and 120 are
modified and probably comprise part of the biotin-binding site of the streptavidin
molecule. Unlike avidin, the modification of lysine residues in streptavidin failed
to result in complete loss of biotin-binding activity. The data imply subtle
differences in the fine structure of the respective biotin-binding sites of the two
proteins.

Molecular cloning and nucleotide sequence of the streptavidin gene.

Using synthetic oligonucleotides as probes we have cloned the streptavidin gene
from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis
indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal
peptide region and the 3' and 5' flanking regions of the gene. The deduced
amino acid sequence shows several interrupted blocks of homology with the
amino acid sequence of chicken egg-white avidin. Analysis of the secondary
structure suggests a high content of beta-structure in both proteins and
considerable overall structural similarity between them.

Streptavidin- biotin binding systems are finding widening applications in

biotechnology,medicines and in environmental studies :

1. The streptavidin protein is joined to a prob. When a sample is incubated

with the biotinylated binder, the binder attaches to any available target
molecules. The presence and location of target molecules can be
determined by treating the sample with a streptavidin probe because the
streptavidin binds to the biotin on he biotinylated binder, and the probe is
then visualized. This detection system is employed in a wide variety of
biotechnological applications, including use as a non-radioactive probe in
hybridization studies and as a critical component in biosensors for a wide
range of environmental monitoring and clinical applications.

Hydrogen bonding network in the

streptavidin-biotin binding site
2. Applications of Streptavidin- biotin binding systems

1. in diagnostics
2. in signal amplification
3. in blotting techniques
4. in immunoassay
5. in bioaffinity sensor
6. in gene probes
7. in chromosome mapping
8. in isolation studies
9. in affinity chromatography
10. in affinity precipitation
11. in immobilizing agents
12. in enzyme reactor systems
13. in selective retrieval
14. in selective elimination
15. in phage –display technology
16. in hybridoma technology
17. in epitope mapping
18. in cell separation
19. in flow cytometry
 20. in fusogenic agent
21. in monolayer technology
22. in affinity perturbation
23. in pathological probe
24. in affinity therapy
25. in drug delivery
26. in imaging
27. in affitnity targeting
28. in cross-linking agents
29. in cytological probes
30. in electron microscopy
31. in fluorescence microscopy
32. in light microscopy
33. in histochemistry
34. in localization studies
35. in affinity cytochemistry

• Streptavidin Agarose For affinity chromatography and

immunoprecipitation of biotinylated

Conjugation Streptavidin is covalently linked to crosslinked agarose beads via a

15-atom hydrophilic spacer arm to produce streptavidin agarose. The specially
designed spacer arm reduces nonspecific binding and ensures optimal binding of
biotinylated molecules. Streptavidin is bound to a final concentration of 2-3 mg of
streptavidin per ml of packed gel.

Applications The streptavidin agarose is suitable for use in the following

applications:
 Affinity chromatography to isolate and purify
biotinylated molecules
Immunoprecipitation (e.g. immunoprecipitation using antibodies which have a
low affinity for protein A including some mouse and rat monoclonal antibodies

A streptavidin-biotin binding system that minimizes blocking by

endogenous biotin.
Pretargeted radioimmunotherapy specifically targets radiation to tumors using
antibody-streptavidin conjugates followed by radiolabeled biotin. A potential
barrier to this cancer therapy is the presence of endogenous biotin in serum,
which can block the biotin-binding sites of the antibody-streptavidin conjugate
before the administration of radiolabeled biotin. Serum-derived biotin can also be
problematic in clinical diagnostic applications. Due to the extremely slow
dissociation of the biotin-streptavidin complex, this endogenous biotin can
irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic
efficacy, as well as reduce sensitivity in diagnostic assays. We tested a
streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than
wild type streptavidin, and three bivalent bis-biotin constructs as replacements
for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics.
Biotin dimers were engineered with certain parameters including water solubility,
biotinidase resistance, and linker lengths long enough to span the distance
between two biotin-binding sites of streptavidin. The bivalent biotins were
compared to biotin in exchange, retention, and off-rate assays. The faster off-
rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin
dimers. In fluorescent competition experiments, the biotin dimer ligands
displayed high avidity binding and essentially irreversible retention with SAv-
Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was
4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings
strongly suggest that employing a mutant streptavidin in concert with a bivalent
biotin can mitigate the deleterious impact of endogenous biotin, by allowing
exchange of bound biotin and retention of the biotin dimer carriers.

• Biotin Conjugated Proteins and Enzymes

Biotinylated HRP, AP, FITC and other molecules for signal amplification
and controls in avidin-biotin methods.
Biotinylated HRP, AP, FITC and other molecules for signal amplification and
controls in avidin-biotin methods : Thermo Scientific Pierce Biotinylated Proteins
include biotin-labeled horseradish peroxidase (B-HRP), alkaline phosphatase (B-
AP) and fluorescein (B-FITC) for use as controls or signal amplification in IHC via
avidin-biotin complex (ABC) techniques.

Biotinylated HRP and biotinylated AP are most commonly used in

immunohistochemistry (IHC) to amplify the signal of biotinylated primary
antibodies using the ABC staining method. Biotinylated Fluorescein (FITC)
cannot be used to polymerize and amplify the signal to the same degree as
biotinylated enzymes because each fluor molecule is tagged with only one biotin
molecule. However, the fluorescent variant of traditional ABC staining is possible
with this unique molecule.

• Use of a sensitive EnVision +-based detection system for Western

blotting: avoidance of streptavidin binding to endogenous biotin and
biotin-containing proteins in kidney and other tissues

Western blotting remains a central technique in confirming identities of proteins,

their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin
system is often used as an amplification step to increase sensitivity but in some
tissues such as kidney, "nonspecific" interactions may be a problem due to high
levels of endogenous biotin-containing proteins. The EnVision system,
developed for immunohistochemical applications, relies on binding of a polymeric
conjugate consisting of up to 100 peroxidase molecules and 20 secondary
antibody molecules linked directly to an activated dextran backbone, to the
primary antibody. This study demonstrates that it is also a viable and sensitive
alternative detection system in Western blotting applications.

• Biotinylated Anti-Streptavidin

Anti-Streptavidin is produced by hyperimmunizing goats with pure streptavidin

and selecting out high affinity, specific antibody by affinity chromatography on
specially prepared agarose bound streptavidin. Anti-Streptavidin and Anti-
Streptavidin conjugates can be used to localize streptavidin or to amplify the
signal produced by streptavidin conjugates. Biotinylated Anti-Streptavidin has the
unusual property of binding to streptavidin either through its antigen binding site
or through its biotin residues. This product is produced by optimally biotin-
labeling our affinity-purified Anti-Streptavidin. By following a fluorochrome
conjugated Streptavidin with Biotinylated Anti-Streptavidin and adding a second
layer of fluorochrome conjugated Streptavidin, the fluorescent signal can be
amplified. This product can be used in tissue staining, chromosome mapping, or
in cell sorter analysis. In addition, Biotinylated Anti-Streptavidin is considered the
industry standard for microarray applications

Step 1: Add Fluorescent Streptavidin

Step 2: Add Biotinylated Anti-Streptavidin
Step 3: Add Fluorescent Streptavidin

• Improved streptavidin compounds for use as research reagents in

diagnostics and drug discovery
The invention relates to novel and improved streptavidin compounds which are
used for diagnostic reagents and drug discovery. The invention includes specific
mutants of Streptomyces avidini, which produce streptavidin. These mutants
provide for novel streptavidin proteins having altered physical properties such as
stability and increased or decreased affinity for binding biotin and other ligands.
The invention overcomes the problems and disadvantages associated with
current utility for streptavidin-biotin complexes and provide a novel streptavidin
with reduced affinity for biotin and methods for utilizing reduced affinity
streptavidin in detection and isolation of biomolecules. This invention will serve
an important need in the diagnostic test, drug discovery and bimolecular
purification market place.

Reduced Affinity Streptavidin

This invention relates to composition and methods for using a mutated
streptavidin protein that has reduced affinity for biotin. Reduced affinity allows for
the use of the streptavidin-biotin coupling systems for detection and isolation
systems wherein it is necessary to remove one or the other of the binding
partners. Such systems are useful for the purification of functional proteins and
viable cells. The invention also relates to nucleic acids which encode reduced
affinity streptavidin protein and to recombinant cells which contain these nucleic
acids. The invention overcomes the problems and disadvantages associated
with current strategies and designs and provide a streptavidin protein with
reduced affinity for biotin and methods fro utilizing reduced affinity streptavidin in
detection and isolation.

Enhanced Affinity Streptavidin or Multiflavor Streptavidin

Compounds and methods are described for producing streptavidin mutants with
greater affinity for biotin substitutes than for biotin. The compounds and methods
of the present invention are particularly useful where levels of endogenous biotin
are present in the system, precluding the use of the standard biotin-avidin
approach. In addition, it is contemplated that the streptavidin-biotin system can
be used as a model to test if the contacts that exist between a protein and a
ligand can serve as the starting point to genetically engineer the protein to
develop a high specificity for another ligand. The strategy is contemplated to be
useful to develop a receptor for a molecule without a known receptor when
phage-display methodologies cannot be employed, such as in the case of a
multi-chain protein, for the discovery of new drugs and diagnostic reagents, or in
applications where the use of one molecule is well-suited for a project but the
other one is not.

Modified Dimeric Streptavidins

This invention relates to recombinant modified single-chain dimeric streptavidin
proteins having two functional biotin binding sites, and to recombinant single-
chain dimeric streptavidin proteins. These proteins have an altered affinity for
binding biotin, particularly an enhanced affinity to bind biotin-4fluorescein. The
modified streptavidin molecules can be used in analysis or composite separation
either alone or in combination with ordinatry streptavidin-biotin systems to
visualize and/or separate composites and molecules.

Applications: Many potential applications, including

1. Reduced affinity streptavidins useful for purification and isolation of
unstable biotinylated targets in which the captured targets must be
released in functional form maintaining their structures and
activities
2. Streptavidin fusion proteins can be used in immunological
applications, such as purification of antibodies, host- and subclass-
specific detection of antibodies and detection of biological materials
through their antibodies.

Immobilization of Nucleic Acids Using Biotin-Strept(avidin) Systems

There are several advantages for using biotin-streptavidin/avidin (strept(avi-din))
systems to immobilize nucleic acids and other molecules. These include the
essential irreversible, but not covalent, binding of biotin to strept(avidin), the ease
of biotinylating a large number of molecules without interfering with their function
or the binding of biotin by strept(avidin), and the stability of strept(avidin)
especially when bound with biotin. Another advantage of the biotin-strept(avidin)
system is that it can be used for rapid prototyping to test a large number of
protocols and molecules. The basic characteristics of the biotin-strept(avidin) are
unique, although many of the approaches for immobilizing reagents with such
systems are not unique. Here, biotin/strept(avidin) immobilizations systems are
reviewed with an emphasis on nucleic acid applications.

• USE OF STREPTAVIDIN- ZAP

The first step toward using Streptavidin-ZAP is to biotinylate the targeting

molecule. Advanced Targeting Systems offers biotinylation services if needed.
The biotinylated molecule and Streptavidin-ZAP are then combined in equimolar
concentrations. The streptavidin-biotin binding happens very quickly, little pre-
incubation time is needed (30 minutes recommended) before the complex can
be used. Once the complex is prepared it can be applied to the experimental
system. One effective assay for screening targeting molecules is a cytotoxicity
assay. Evidence of cell death in vitro can be demonstrated in 72 hours. Results
of Streptavidin-ZAP use in a cytotoxicity assay .In this assay, three reagents
were added to cells: unconjugated saporin, a direct conjugate of IB4 to saporin
(IB4-SAP), and Streptavidin-ZAP combined with biotinylated IB4. This assay
compares the effectiveness of a direct conjugate with a secondary conjugate
complex. As can be seen, this assay demonstrates that the results with the
Streptavidin-ZAP/IB4 complex are highly reliable indicators of how effective a
direct conjugate using IB4 would be. Thus, once you screen your potential
targeting molecules with Streptavidin-ZAP, you can have a high level of
confidence in having a direct conjugate produced

Current Research

Magnetic Chains and Microfluidics

Poly-ethylene-glycol Linked Chains of Magnetic Colloidal Particles:

Mechanics and Applications
Paramagnetic particles have the unique ability to aggregate into reversible linear
chains under the influence of an external magnetic field. We create permanently
linked chains by crosslinking 1mm particles using streptavidin-biotin chemistry.
Streptavidin is a tetrameric protein with a high affinity for the molecule biotin.
Streptavidin-coated microspheres are placed in a flow cell and a magnetic field is
applied, causing the particles to form chains. Then a solution of polymeric linkers
of bis-biotin-polyethylene glycol molecules is added in the presence of the field.
These linked chains remain responsive to the magnetic field, allowing their
orientation and flexibility to be controlled by directing the field and changing the
field strength. Cross-linked paramagnetic particles form flexible chains which can
move and bend freely. In the presence of a magnetic field, the chains stiffen in
the direction of the magnetic field.

Magnetic Resonance Molecular Imaging of the HER-2/neu Receptor

The HER-2/neu receptor is a member of the epidermal growth factor family and
is amplified in multiple cancers. It is under intense investigation both as a
prognostic marker and for therapy, using monoclonal antibodies targeted against
the receptor. We have developed a novel two-component gadolinium-based MR
contrast agent to image the HER-2/neu receptor. Positive T1 contrast in MR
images was generated by the specific binding of avidin-gadolinium complexes to
tumor cells prelabeled with a biotinylated anti-HER-2/neu antibody. Significant
intensity enhancement was observed in HER-2/neu-expressing cell lines and in
vivo in a breast cancer model. Potential applications of this approach may
include determination of the HER-2/neu status for prognosis and for selecting
tumors for monoclonal antibody therapy

Streptavidin binding to biotinylated lipid layers on solid supports. A

neutron reflection and surface plasmon optical study
Neutron reflection and surface plasmon optical experiments have been
performed to evaluate structural data of the interfacial binding reaction between
the protein streptavidin and a solid-supported lipid monolayer partly
functionalized by biotin moieties. Since both experimental techniques operate in
a total internal reflection geometry at a substrate/solution interface, identical
sample architectures allow for a direct comparison between the results obtained
with these two recently developed methods. It is found that a monomolecular
layer of dipalmitoyllecithin doped with 5 mol% of a biotinylated-
phosphatidylethanolamine shows a thickness of d1 approximately (3.4 +/- 0.5)
nm. Binding of streptavidin to the biotin groups results in an overall layer
thickness of d = (5.9 + 0.5) nm that demonstrates the formation of a well-ordered
protein monolayer with the (biotin+spacer) units of the functionalized lipids being
fully embedded into the binding pocket of the proteins. It is demonstrated by
model calculations that a more detailed picture of the internal structure of this
supramolecular assembly can only be obtained if one uses deuterated lipid
molecules, thus generating a high contrast between individual layers.

Screening for the breast cancer gene (BRCA1) using a biochip system and
molecular beacon probes immobilized on solid surfaces

This describe the use of a biochip based on complementary metal oxide

semiconductor (CMOS) technology for detection of specific genetic sequences
using molecular beacons (MB) immobilized on solid surfaces as probes. The
applicability of this miniature detection system for screening for the BRCA1 gene
is evaluated using MB probes, designed especially for the BRCA1 gene. MB
probes are immobilized on a zeta-probe membrane by biotin-streptavidin
immobilization. Two immobilization strategies are investigated to obtain optimal
assay sensitivity. The MB is immobilized by manual spotting on zeta-probe
membrane surfaces with the use of a custom-made stamping system. The
detection of the BRCA1 gene using an MB probe is successfully demonstrated
and expands the use of the CMOS biochip for medical applications.

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