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 characteristicsStrain 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 onGauze I grayish pink NDISP medium 2 N one Grayish yellowish pink toISP medium 3 N one light grayish reddishISP medium 4 N one brown on ISP 2, 3, 4, 5ISP medium 5 beige

Colour of substrate mycelium on:Gauze I pale yellow NDISP medium 2 N one C olorless or pale yellowISP medium 3 N one to light yellowish brownISP medium 4 white on ISP 2, 3, 4, 5ISP medium 5 beige

Colour of soluble pigment on:Gauze I yellow N DISP medium 2 N one N oneISP medium 3 N one N oneISP medium 4 N one N oneISP medium 5 N one N one

Melanin production on tyrosin agar N one NoneMaximum NaCl tolerance (%, w/v) 7 ND

Growth at: *4°C + ND28°C ++ ++

Growth on sole carbon sources(1%, w/v):Adonitol - N DArabinose - -Cellobiose +/- N DFructose + +D-galactose +/- N DD-glucose + +Innulin - N DD-lactose - N DTrehalose +/- N DMannitol +/- -D-mannose - NDD-melibiose +/- NDRaffinose +/- -L-rhamnose - +/-Salicin +/- NDSucrose - +/-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 analysisAmplification 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 SB9with primer combination 243F/A3R. Lane 1: GeneRuler - 1 kb DNA Ladder(Fermentas); Lane 2: SB9; Lane 3: positive PCR control (Streptomycesrimosus)

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 ~10-14 mol/L, making it one of the strongest non-covalent interactions known in nature. It is used extensively in molecular biology and bionanotechnology as, in addition to the high affinity, biotin-binding is resistant to extremes of pH, temperature, organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton) and proteolytic enzymes.

Structure

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 inbiotechnology,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 diagnostics2. in signal amplification3. in blotting techniques4. in immunoassay5. in bioaffinity sensor6. in gene probes7. in chromosome mapping8. in isolation studies9. in affinity chromatography 10. in affinity precipitation11. in immobilizing agents12. in enzyme reactor systems13. in selective retrieval14. in selective elimination15. in phage –display technology16. in hybridoma technology17. in epitope mapping18. in cell separation19. in flow cytometry

20. in fusogenic agent21. in monolayer technology22. in affinity perturbation23. in pathological probe24. in affinity therapy 25. in drug delivery 26. in imaging27. in affitnity targeting28. in cross-linking agents29. in cytological probes30. in electron microscopy31. in fluorescence microscopy32. in light microscopy 33. in histochemistry34. in localization studies35. 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 followingapplications:Affinity chromatography to isolate and purifybiotinylated 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 StreptavidinStep 2: Add Biotinylated Anti-StreptavidinStep 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 mutantsprovide 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 withcurrent 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 StreptavidinThis 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 StreptavidinCompounds 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 biotinare 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 StreptavidinsThis 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 forbinding 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, including1. 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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