# Patent

## Title
Monomeric streptavidin muteins

## Abstract

The invention includes a streptavidin mutein having at least one mutation chosen to cause one or more of steric hindrance, charge repulsion, or improvement of solubility by changing interfacial hydrophobic residues to less hydrophobic or hydrophilic residues. The mutein may exist in monomeric form even in the presence of biotin, and reversibly binds to biotin. The invention also includes polynucleotides encoding such muteins, expression systems and host cells for producing such muteins. The invention also includes a method of capturing biotinylated molecules using the muteins of the invention.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a division of U.S. patent application Ser. No. 11/307,576 filed on Feb. 13, 2006 entitled “Monomeric Streptavidin Muteins”, now U.S. Pat. No. 7,265,205, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to monomeric streptavidin muteins. In particular, the invention relates to production of an engineered monomeric streptavidin for use in the capture and/or purification of biotinylated proteins.
BACKGROUND
High throughput systems have been used to generate affinity tagged recombinant proteins to assist in their immobilization and purification, to study their function and structure. Although a number of tags have been developed, those that can be used efficiently for both purification and immobilization are limited; and biotinylation offers potential for both tasks. The tight binding of biotin to avidin and streptavidin results in capturing of biotinylated biomolecules. The availability of a form of streptavidin in appropriate quantities that binds reversibly, such that it can be used for capture and/or purification of biotinylated proteins in acceptable yield would significantly extend potential applications of biotin-streptavidin technology (Bayer, 1990; Bashir, 2001; Niemeyer, 1999; Schechter, 1999; Casalini, 1997).
Monomeric avidin may be obtained by immobilizing tetrameric avidin molecules on to a matrix followed by denaturation and renaturation (Henrikson, 1979; Kohanski, 1990). The process for denaturation and renaturation is fairly expensive with loss of non-immobilized avidin subunits and the consumption of a large quantity of expensive reagents in the denaturation step. Furthermore, the high stability of avidin and the strong interfacial interactions between avidin subunits make complete denaturation challenging. Any remaining tetrameric avidin molecules in the matrix can lead to reduced recovery of biotinylated molecules during elution (Kohanski, 1990).
Although streptavidin and avidin have similar three-dimensional structures and biotin binding properties, development of monomeric streptavidin is considered more challenging for at least two reasons. First, streptavidin has stronger subunit interfacial interactions than avidin (Bayer, 1996, Waner, 2004). It is more difficult to weaken this strong interface interaction. Second, monomerization of streptavidin may result in the surface exposure of hydrophobic residues, which normally would be buried at the interface in tetrameric streptavidin. This can potentially affect the solubility of the monomeric streptavidin and lead to re-association of the monomers. Solubility is less of an issue for avidin, which is a glycosylated protein with a carbohydrate chain in each of the avidin subunits.
However, engineered versions of monomeric avidin and streptavidin (Qureshi, 2001; Qureshi, 2002) have been developed. These mutations led to weak H-bonding interactions between streptavidin and biotin, and also affected subunit interactions at the interface. However, the low affinity of this mutein towards biotin (Kd=1.7×10−6M) makes it less than ideal.
Streptavidin is a homo-tetrameric molecule with a biotin binding site in each subunit (Green, 1990). The three dimensional structure of streptavidin (Hendrickson, 1989; Weber, 1989) suggests that a complete biotin binding pocket in each subunit requires the contribution of a tryptophan residue (Trp-120 for streptavidin and Trp-110 for avidin) from an adjacent subunit. Site-directed mutagenesis studies also demonstrate the importance of this residue for tight biotin binding and subunit communications (Freitag, 1998; Sano, 1995; Laitinen, 2001). In the case of avidin, the first generation of engineered monomeric avidin can exist in the monomeric state only in the absence of biotin (Laitinen, 2001). This problem has been solved by the development of a monomeric avidin (Laitinen, 2003), which carries two mutations (N54A, W110K). Structural alignment of avidin and streptavidin indicates that these two residues correspond to D61 and W120 in streptavidin (Livnah, 2003). However, a streptavidin mutein (AK mutein) which carries the corresponding double mutations (D61A, W120K) does not become monomeric (Wu, 2005). Although AK mutein shows reversible biotin binding property and monomeric behavior on the SDS-polyacrylamide gel, it clearly exists in the oligomeric state in solution.
The challenges in producing a monomeric streptavidin mutein, which has useful biotin binding properties, have not yet been completely addressed. There is a need in the art for a streptavidin mutein that may be produced in a soluble and functional form and that reversibly binds to biotinylated biomolecules, which can then be used for capture and/or purification of biotinylated biomolecules.
SUMMARY OF THE INVENTION
The present invention relates to the engineering and production of a monomeric streptavidin, referred to herein as a mutein. In a preferred embodiment, the invention relates to the engineering and production of monomeric streptavidin mutein using a suitable expression system and host cell such as B. subtilis or E. coli. In one embodiment, the monomeric streptavidin mutein includes site-specific mutation or mutations that allow for formation of the monomer and reversibly bind to biotin.
The mutein may be used for the capture and purification of biomolecules. In a preferred embodiment, the engineered monomeric streptavidin reversibly interacts with biotinylated biomolecules and assist with their capture and purification.
The inventors have determined that a mutation of a residue located on a rigid surface, to introduce either charge repulsion and steric hindrance, or both, at the interface, can be effective in developing monomeric streptavidin. The efficiency of streptavidin monomerization may be further enhanced by introducing a second mutation (M2). In a preferred embodiment, further mutations may be introduced to ensure that the streptavidin mutein will stably remain in the monomeric state and in solution.
Therefore, in one aspect, the invention may comprise a streptavidin mutein comprising at least one mutation chosen to cause one or more of steric hindrance, charge repulsion, or improvement of solubility by changing interfacial hydrophobic residues to less hydrophobic or hydrophilic residues. In one embodiment, the mutein comprises an amino acid sequence comprising a mutation wherein Thr76 is substituted with a charged amino acid. The mutein may further comprise at least one mutation of a hydrophobic interfacial amino acid residue to a less hydrophobic, charged or hydrophilic residue. In one embodiment, at least one mutation is selected from the group consisting of:
(a) Val125 substituted with a charged or hydrophilic amino acid;
(b) Val55 substituted with a charged or hydrophilic amino acid; and
(c) Leu109 substituted with a charged or hydrophilic amino acid.
In a preferred embodiment, the mutein comprises at least four mutations where Thr76 is substituted with Arg, Val125 is substituted with Arg, Val55 is substituted with Thr; and Leu109 is substituted with Thr. In another aspect, the invention may comprise a polynucleotide which encodes for a streptavidin mutein as described herein, an expression vector comprising such polynucleotides, or a host cell comprising such an expression vector. The host cell may comprise B. subtilis or E. coli. 
In another aspect, the invention may comprise a method of isolating a biotinylated molecule from a sample, comprising the steps of binding a streptavidin mutein as described herein to a solid support to form a matrix, passing the sample over the support, and eluting the biotinylated molecule. The solid support matrix may then be regenerated for reuse.



BRIEF DESCRIPTION OF THE DRAWINGS
Exemplary embodiments of the invention may be described with reference to the following figures.
FIG. 1: Construction of the pV55T-T76R-L109T-V125R expression vector for the production of a streptavidin mutein M4. The restriction sites used in construction are indicated. The 164-bp BamHI/ScaI fragment bearing T76R and L109T mutations was generated through PCR. The ScaI/NheI fragment carrying the V125R mutation was from pV125R. P43, P43 promoter; SacB SP, B. subtilis levansucrase signal peptide; Tcry, the transcription terminator from the Bacillus thuringiensis crystal toxin gene; SAV, synthetic streptavidin gene. Asterisk, closed circle, open circle and closed triangle represent V55T, T76R, L109T and V125R mutations, respectively.
FIG. 2: Structure of tetrameric streptavidin and critical interface residues selected for mutagenesis. FIG. 2A. Interfaces of tetrameric streptavidin. Subunit A forms three subunit contact interfaces with other subunits. These interfaces include A/B, A/C and A/D. Subunits A, B, C and D are highlighted in red, orange, yellow and green, respectively. FIG. 2B. Local environment of Thr-76 in subunit A. This shows the interfacial interaction between subunits A and B. Thr-76 in subunit A is close to both Thr-76 and Arg-59 in subunit B. Thr-75 and Arg-59 in subunit A are highlighted in red and pink, respectively. Thr-76 and Arg-59 in subunit B are in dark and bright yellow, respectively. FIG. 2C. Local environment of Val-125 in subunit A. This shows the interfacial interaction between subunit A and D. Val-125 highlighted in red fits into a pocket formed by Val-125 (green) and Thr-123 (green) from subunit D. FIG. 2D. Local environment of Val-125 in subunit A. Val-55 (red) in subunit A is close to Arg-59 (yellow) in subunit B.
FIG. 3: Western blot analysis of cell culture supernatants from B. subtilis strains producing streptavidin muteins carrying a single mutation. 15 μl of non-boiled sample of culture supernatant was loaded to each lane. Samples on the left set were collected from B. subtilis strains cultured in super-rich medium without additional supplement of biotin. Samples on the right set were collected from B. subtilis strains cultured in super-rich medium with additional supplementation of biotin (20 μM). The blot was probed with polyclonal antibodies against streptavidin and developed using 4-chloro-1-napthol (Bio-Rad) as the colour development agent. Culture supernatant from WB800 [pWB705HM] (Wong et al., 1995), which did not produce any streptavidin served as the negative control. M, molecular weight markers; wt, wild type streptavidin.
FIG. 4: Analysis of culture supernatants from B. subtilis strains producing streptavidin muteins carrying different combinations of mutations. All these strains were cultivated in super-rich medium supplemented with biotin (20 μM). The 15 μl of non-boiled samples of culture supernatants were loaded. FIG. 4A. Coomassie blue stained SDS-polyacrylamide gel. Bands corresponding to tetrameric and monomeric streptavidin are marked by an asterisk and an arrowhead, respectively. FIG. 4B. Western blot probed with polyclonal antibodies against streptavidin. M, molecular weight markers; wt, wild type streptavidin; AK, mutein (D61A, W120K); M1, mutein (T76R); M2, mutein (T76R, V125R); M4, mutein (T76R, V125R, V55T, L109T); C, negative control.
FIG. 5: Purification of the M4 streptavidin mutein using biotin-agarose. M4 mutein partially purified by Macro S column chromatography (PPF) was loaded on a biotin-agarose column. M, molecular weight markers; FT, column flow-through; W, pooled washing fractions; EF1-EF3, eluted fractions.
FIG. 6: Gel filtration profiles of streptavidin muteins. FIG. 6A. Determination of apparent molecular mass of streptavidin muteins in the absence of biotin using Bio-Prep SE 100/17 gel filtration column. Bovine gamma-globulin (158 kDa), bovine serum albumin (66 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B-12 (1.35 kDa) were used as molecular mass markers (closed circles) to calibrate the column. The logarithm of molecular mass was plotted versus Kav (Kav=(Ve−Vo)/(Vt−Vo); Ve, elution volume; Vo, void volume; Vt, bed volume). Positions of wild-type streptavidin (wt) and streptavidin muteins are represented by solid squares. FIG. 6B. Elution profile of M4 mutein (loaded at 2 mg/ml) on the gel filtration column.
FIG. 7: Determination of the monomeric or oligomeric states of wild-type streptavidin and its muteins. Pictures show the Coomassie blue stained SDS-polyacrylamide gel. FIG. 7A. Proteinase K digestion. FIG. 7B. Cross-linking study using sulfo-EGS as the cross-linker. All samples were boiled prior to loading. M, molecular weight markers; wt, wild type streptavidin; L, lysozyme. Numbering represents streptavidin molecules in monomeric (1), dimeric (2) and oligomeric (3-5) states, respectively.
FIG. 8: Kinetic parameters for the interaction between M4 streptavidin mutein and biotin as monitored by BIAcore biosensor. A. On-rate (ka) determination. ka is derived from association analysis. kd is the slope of the plot of ln(Ro/Rt) versus (t−to) where Ro is the response at an arbitrary starting time to; Rt is the response at time t.
FIG. 9: Coomassie blue-stained SDS-polyacrylamide gel showing intracellular production of monomeric streptavidin in E. coli. Samples loaded were not boiled. Lane M, molecular weight markers. Lane 1 and 2, E. coli BL21[pET-SAVM4]. Lanes 3 and 4, negative control, E. coli BL21[pET29b]. S, soluble fraction; I, insoluble fraction. Asterisk marks the position of the M4 mutein.
FIG. 10: Purification of M4 mutein using biotin-agarose column. Lane M, molecular weight markers. Lane 1, soluble fraction of crude lysate. Lane 2, column flow-through. Lanes 3-5, eluted fractions. Asterisk marks the position of the M4 mutein.
FIG. 11: Coomassie blue-stained SDS-polyacrylamide gel showing purification of biotinylated proteins using M4 affinity column. FIG. 11A Purification of biotinylated cytochrome c. Asterisk marks the position of the cytochrome c. FIG. 11B Purification of biotinylated MBP-AviTag. Asterisk marks the position of the MBP-aviTag. Lane M, molecular weight markers. Lane 1, crude sample containing B. subtilis cell lysate and biotinylated protein. Lane 2, column flow-through. Lane 3, one-column wash. Lanes 4 and 5, eluted fractions.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides for a streptavidin mutein having useful properties. As used herein, a “mutein” means a genetically engineered protein arising as a result of a laboratory-induced mutation. When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.
As used herein, “streptavidin” refers to wild-type protein, and includes naturally occurring variants thereof. The reference to amino acid positions herein refers to the amino acids in the mature streptavidin protein, from which the signal sequence has been removed, the sequence of which is reproduced below, and obtained from GenBank (National Center for Biotechnology Information, USA), for example as Accession #X03591 or #AF283893.







TABLE 1





SEQ ID. NO: 1 (mature monomeric streptavidin subunit protein sequence)


















        10         20         30         40         50         60




         |          |          |          |          |          |


  1
DPSKDSKAQV SAAEAGITGT WYNQLGSTFI VTAGADGALT GTYESAVGNA ESRYVLTGRY


 


 61
DSAPATDGSG TALGWTVAWK NNYRNAHSAT TWSGQYVGGA EARINTQWLL TSGTTEANAW


 


121
KSTLVGHDTF TKVKPSAASI DAAKKAGVNN GNPLDAVQQ









Reference herein to the streptavidin nucleic acid or polynucleotide, and nucleotide positions therein, is a reference to any nucleic acid encoding streptavidin, and includes, for example the wild-type nucleic acid (SEQ ID NO: 2) or a synthetic nucleic acid (SEQ ID NO:3)








TABLE 2







SEQ ID. NO: 2











        10         20         30         40         50         60




         |           |           |          |          |          |


  1
CCCTCCGTCC CCGCCGGGCA ACAACTAGGG AGTATTTTTC GTGTCTCACA TGCGCAAGAT


 


 61
CGTCGTTGCA GCCATCGCCG TTTCCCTCAC CACGGTCTCG ATTACGGCCA GCGCTTCGGC


 


121
AGACCCCTCC AAGGACTCGA AGGCCCAGGT CTCGGCCGCC GAGGCCGGCA TCACCGGCAC


 


181
CTGGTACAAC CAGCTCGGCT CGACCTTCAT CGTGACCGCG GGCGCCGACG GCGCCCTGAC


 


241
CGGAACCTAC GAGTCGGCCG TCGGCAACGC CGAGAGCCGC TACGTCCTGA CCGGTCGTTA


 


301
CGACAGCGCC CCGGCCACCG ACGGCAGCGG CACCGCCCTC GGTTGGACGG TGGCCTGGAA


 


361
GAATAACTAC CGCAACGCCC ACTCCGCGAC CACGTGGAGC GGCCAGTACG TCGGCGGCGC


 


421
CGAGGCGAGG ATCAACACCC AGTGGCTGCT GACCTCCGGC ACCACCGAGG CCAACGCCTG


 


481
GAAGTCCACG CTGGTCGGCC ACGACACCTT CACCAAGGTG AAGCCGTCCG CCGCCTCCAT


 


541
CGACGCGGCG AAGAAGGCCG GCGTCAACAA CGGCAACCCG CTCGACGCCG TTCAGCAGTA


 


601
GTCGCGTCCC GGCACCGGCG GGTGCCGGGA CCTCGGCC









In the nucleotide sequence for the wild type streptavidin subunit, the precursor sequence is between nucleotides 50-598. The coding sequence for the mature wild type streptavidin subunit is between nucleotides 122-598. The translation stop codon is located between nucleotides 599-601.)
As used herein, “affinity” refers to the dissociation constant (Kd) of a complex between a mutein or streptavidin, and biotin, as determined by standard methods known to those skilled in the art, an example of which is disclosed in the Examples herein. As is known in the art, Kd is related to the association constant Ka, being the inverse thereof.
As used herein, “lower binding affinity” means a decrease in the binding affinity relative to streptavidin, which can be measured as an increase in Kd or, alternatively, a decrease in Ka. The lower binding affinity can result from a change in one or both of kon (the “on rate”, or “association rate constant”, or ka) and koff (the “off rate”, or “dissociation rate constant, or kd).
As used herein, “monomeric form” or “monomer” means a streptavidin or mutein subunit that is not in the form of a complex with another streptavidin or mutein subunit, or that does not form a complex with another streptavidin or mutein subunit.
As used herein, “tetrameric form” or “tetramer” means a protein complex that is comprised of four streptavidin and/or mutein subunits, and includes complexes in which one or more of the four subunits are different from one another.
As used herein, “subunit” means the protein that is generated from a streptavidin or mutein gene. “Subunit” is used interchangeably with “monomer”.
As used herein, “biotin” means biotin in its free form as well as in the form of biotinylated substances such as biotinylated nucleic acids, carbohydrates, lipids, peptides or polypeptides, biotin analogues and biotin derivatives such as iminobiotin, desthiobiotin and streptavidin affinity peptides.
As used herein, amino acids shall be referred to their commonly accepted abbreviations as well as their numerical position as indicated by SEQ ID NO: 1. As may be appreciated, if amino acid substitutions or deletions are made in mutein of this invention, the numerical position of a particular amino acid in the mutein may be altered.
As shown in the FIG. 2, the interface between subunits A and B (and between C and D) has the most extensive subunit interactions. The interfacial contact area between A and B is  ̃1,557 Å2 with 17H-bonding interactions, two salt bridges and numerous van der Waal interactions. The interface contact between A and D is also extensive with a contact area of 525 Å2 and two interfacial H-bonding interactions. The weakest interface interaction is between subunits A and C with an interfacial contact area of 171 Å2.
The present invention incorporates mutations designed to introduce charge repulsion, steric hindrance, or hydrophilicity, or combinations thereof, in order to achieve useful properties. As proteins are known to have structural plasticity, it is preferred to select interfacial residues located on a rigid surface to maximize the effects of charge repulsion and steric hindrance. Since streptavidin subunit forms an eight-antiparallel stranded β-barrel structure (Weber, 1989; Hendrickson, 1989), the selected residues are preferably located on the β-strands rather than in the loop regions. Furthermore, in preferred embodiments, the selected residue in one subunit should be located very close to the equivalent residue or a charged residue in another subunit at the interface. The exposure of the AB (or CD) interface will expose hydrophobic residues. Based on the above criteria and upon examination of interfacial residues (FIGS. 2B-D), four amino acids in particular, Thr-76, Val-125, Val-55, and Leu-109 meet at least one criterion. Hence, in a preferred embodiment, these four are preferred candidates for mutagenesis.
The monomeric muteins of streptavidin may be made using methods known to those skilled in the art, for example by cassette mutagenesis of streptavidin and expression of the mutated streptavidin gene in an appropriate host, an example of which is included in the Examples herein. As is apparent, different PCR primers than those used in the Examples herein may be used, and will likely be needed, if one is using as a PCR template a nucleic acid for streptavidin that is different than the synthetic ssav gene, for example the wild-type gene for streptavidin, or another gene that codes for streptavidin. Those skilled in the art know how to design alternative primers for use in making the mutein herein. The affinity of the monomeric mutein of streptavidin for biotin can be measured using techniques known to those skilled in the art, for example by using a surface plasmon resonance (SPR) based BIAcoreXTM biosensor, as disclosed in the Examples herein.
Streptavidin muteins carrying a single amino-acid change at the selected site may be produced in their soluble form by B. subtilis via secretion. Analysis of non-boiled culture supernatants by SDS-PAGE offers a quick screen for the mutation effect (Qureshi, 2001). Weaker subunit interaction would result in a higher percentage of the sample in the monomeric state on the SDS-polyacrylamide gel. Since biotin can strengthen subunit interaction, samples were analyzed in the presence or absence of biotin (Laitinen, 2001; Gonzalez, 1999).
As demonstrated in the Examples, a single mutation is sufficient to cause a mutein to exist as a monomer, even in the presence of biotin. In one embodiment, the mutation is a substitution of Thr76 (T76). Without being bound to a theory, it is believed that because this residue has a solvent accessible area of zero, exists close to both T76 and Arg-59 in subunit B and is located on a rigid surface of a β-barrel structure, it is an ideal residue to be changed in the present invention. In a preferred embodiment, it is changed to an amino acid with a relatively bulky and charged side chain, such as arginine, lysine or histidine, to achieve the maximal electrostatic repulsion and steric hindrance effects at the subunit interface. In another embodiment, the mutation is a substitution of Val-125, which has a solvent accessible area in subunit A of 1.78%. It has extensive interactions with Leu-109, Trp-120, Thr-123 and Val-125 in subunit D (FIG. 2C), Leu-109 in subunit B and Gln-107 in subunit C. Its conversion to an amino acid with a relatively bulky and charged side chain, such as arginine, lysine or histidine, results in charge repulsion in subunit D and potential steric hindrance for subunits B, C, and D.
When choosing substitutes for muteins of the present invention, charged amino acids may be either positively or negatively charged amino acid residues. Positively charged amino acids may be preferred. In addition to the potential charge repulsion effect, charged amino acids are hydrophilic, which may aid in the solubility of the resulting mutein.
In a preferred embodiment, a second mutation may be added to develop monomeric streptavidin muteins that are more likely to remain in the monomeric state at high streptavidin concentrations, in the presence of biotin, and have excellent reversible biotin binding capability. A double mutant carrying both T76 and V125 mutations is referred to herein as M2. In one example, M2 carries T76R and V125R mutations.
In another preferred embodiment, additional mutations are possible. Preferably, one or more additional interfacial amino acids are changed. A quadruple mutant carrying T76, V125, V55 and L109 mutations is referred to herein as M4. In one example of M4, in addition to the two mutations of M2, two additional interfacial hydrophobic residues Val-55 and Leu-109 are changed to hydrophilic amino acids. Therefore, in one example, M4 carries T76R, V125R, V55T and L109T mutations. Other mutations of interfacial hydrophobic residues to less hydrophobic, or hydrophilic residues are within the scope of the present invention.
As shown in FIG. 4 and Table 4, examples of M2 and M4 exist in monomeric state on the SDS-polyacrylamide gel even in the presence of biotin.
In one example of a M2 mutein, Val-125 has been changed to arginine. Val-55 and Leu-109 may be changed to a more hydrophilic residue, such as threonine, as in a M4 mutein embodiment. Conversion to threonine instead of arginine is preferred because proteins with high pI are known to have non-specific interactions via electrostatic interactions (Marttila, 1998; Marttila, 2000). The calculated pI of M2 (V55T, L109T) is 8.1. If both Val-55 and Leu-109 are converted to arginine, the resulting mutein will have a pI of 9.2. This may increase the chance of charge-related non-specific interactions. The conversion of these residues to negatively charged residues is possible, but not preferred because both Val-55 and Leu-109 are located on the β-strands and negatively charged residues are relatively poor β-strand formers.
M4 mutein, like M2 mutein, exists in the monomeric state at a reasonably high protein concentration (2 mg/ml or more as used in dynamic light scattering study). Both have excellent reversible biotin binding capability as reflected by their on-rate and off-rate for biotin interaction. Both have a moderate pI value of 8.1 so that charge-related non-specific interactions will be minimal. M4 is a preferred embodiment as it displays additional two features. Molecules of M4 mutein are less likely to aggregate in solution. M2 is preferably filtered through a 0.02 μm filter in order to obtain a good signal of the mutein for dynamic light scattering studies because of poor signal detection caused by the presence of small amounts of large aggregates in an unfiltered sample. Good results may be obtained with M4 with an unfiltered sample of similarly prepared M4. Thus, conversion of the two hydrophobic residues (Val-55, Leu-109) to the more hydrophilic threonine residue does assist in minimizing aggregation of the mutein. Also, M4 has a sharp elution profile with its purification using biotin-agarose. Over 95% of the mutein could be readily eluted off from the column using just two column volumes of the eluant, leading to a high rate of protein recovery.
Muteins of the present invention may include additional amino or carboxyl-terminal amino acids, or amino acids interior to the mutein (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed from the engineered protein by cellular enzymes.
Muteins of the present invention may also include insubstantial variants such as those carrying silent substitutions, additions and deletions that do not significantly alter the properties and activities of the mutein.
In another aspect of the invention, the invention comprises polynucleotides that encode the streptavidin muteins described herein. Such polynucleotides may encode the amino acid sequence set out in Table 1 [SEQ ID NO: 1] with one or more of the selected mutations. The polynucleotides of the present invention may include, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
A preferred polynucleotide comprises the nucleotide sequence shown in Table 2 [SEQ ID NO: 2] with mutations which encodes the muteins described herein. Other polynucleotides included in the present invention may encode biologically active variants of the muteins.
Using the information provided herein, such as a polynucleotide sequence set out in Table 2 [SEQ ID NO: 2], the portion of the polynucleotide of the invention encoding streptavidin may be obtained using standard screening and cloning methods. For example, to obtain a polynucleotide fragment comprising some of all of the streptavidin sequence, a Streptomyces species chromosomal DNA library is probed by hybridization with a synthetic radiolabeled oligonucleotide, preferably a 17-mer or longer, that is homologous to the streptavidin sequence. Clones carrying DNA highly homologous to the probe are identified by using stringent hybridization conditions. By sequencing the individual clones identified by hybridization with sequencing primers designed from the sequences in the plasmid or phage DNA from which the library was constructed, it is possible to identify the streptavidin clones. The DNA inserts from several clones can be ligated together to obtain a full-length polynucleotide, if necessary. Suitable techniques for manipulating DNA are described by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).
The invention may provide a coding sequence for a mature protein or a portion thereof, by itself, as well as a coding sequence for a mature protein or a portion thereof in frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotides of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5′ and 3′ sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids.
The term “polynucleotide encoding a polypeptide” as used herein encompasses polynucleotides that include a sequence encoding a mutein of the present invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the engineered protein (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions that also may contain coding and/or non-coding sequences.
Preferred embodiments include polynucleotides encoding proteins that retain substantially the same biological function or activity as a M1, M2 or M4 mutein described herein. Further particularly preferred embodiments are polynucleotides encoding variants of M1, M2 or M4 muteins, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions that do not alter the properties and activities of the mutein.
Other preferred embodiments of this invention may include polynucleotides that hybridize, particularly under stringent conditions, to polynucleotides described herein including polynucleotide sequences encoding the engineered protein. As herein used, the terms “stringent conditions” and “stringent hybridization conditions” mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the MarathonTM technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the “missing” 5′ end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using “nested” primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3′ in the adaptor sequence and a gene specific primer that anneals further 5′ in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5′ primer.
The invention also provides polynucleotides that encode a polypeptide that is the mutein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mutein (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed from the engineered protein by cellular enzymes.
For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it. It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.
In addition to the standard A, G, C, T/U representations for nucleotides, the letter “N” may also be used in describing a nucleotide. “N” means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that, when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode the muteins described herein, the muteins plus a leader sequence (which may be referred to as a preprotein), or a precursor of the mutein, having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the mutein polypeptide, and insubstantial variants of these polypeptides.
The streptavidin muteins of the present invention may be produced at a reasonable level (50-60 mg/liter) in a prokaryotic expression system, such as B. subtilis via secretion (Wu, 2005). In a preferred embodiment, in order to affinity purify the protein using biotin-agarose, the culture supernatant containing the secreted streptavidin mutein is reduced to a manageable volume and freed of biotin contaminants, by dialysis or other means.
In one embodiment, intracellular production in E. coli offers an alternative approach. Toxicity arising from depletion of intracellular biotin by soluble streptavidin (Szafranski, 1997) is not a significant concern since the streptavidin muteins of the present invention have much lower affinity towards biotin. Despite loss of mutein in the insoluble fraction (40-50%), the soluble portion represents a relatively high production yield (around 70 mg/liter) of mutein (FIG. 9). Thus, in one embodiment, with approximately 70% recovery of purified M4 using biotin-agarose chromatography (FIG. 10), 40-50 mg of purified M4 could be obtained from one liter of cell culture.
In order to produce streptavidin mutein intracellularly in E. coli in the soluble form, it is preferred to use the mutein gene encoding the full-length version of streptavidin. Streptavidin and its muteins are traditionally produced intracellularly in E. coli in a truncated form known as core streptavidin (Pahler, 1987; Sano, 1995; Hyre, 2000; Freitag, 1998; Sano, 1995). This is because the core version represents the form of streptavidin that can be crystallized well for X-ray crystallographic studies. It is also one of the shortest versions of streptavidin that retains full biotin binding capability. However, these core streptavidin molecules predominantly form inclusion bodies. The full-length version (159 amino acids—SEQ ID NO: 1) has a 13 amino-acid extension at the N-terminus and a 20 amino-acid extension at the C-terminus (Pahler, 1987; Sano, 1995; Bayer, 1990). The amino acids in these terminal peptides are mainly charged and polar. Although these sequences are not required for biotin binding, their presence clearly helps minimize the formation of inclusion bodies. This is particularly true when streptavidin is overproduced intracellularly. In one embodiment, various solubility partners such as translation initiation factor IF2, maltose binding protein, NusA, and T7 tag may be fused to streptavidin and can improve the intracellular production of streptavidin in its soluble form (Sorensen, 2003; Gallizia, 1998). In many cases, removal of the fusion partner is required to bring streptavidin back to its original size. Therefore, the use of the natural N- and C-terminal segments from streptavidin is preferred to address the solubility problem associated with streptavidin production. The change of three interfacial hydrophobic residues in M4 changed to either charged or hydrophilic residues, results in excellent solubility even at a concentration of 2 mg/ml (Wu, 2005).
In one embodiment, E. coli cells expressing streptavidin muteins may be harvested and disrupted with a French press in a known manner. The crude cell lysate may be separated into soluble and insoluble fractions by centrifugation. The soluble fraction is dialysed to remove any biotin contaminants and monomeric streptavidin in the dialysate may be purified using known techniques, such as on a biotin-agarose column (FIG. 10).
Muteins of the present invention may be purified by techniques well known to those skilled in the art. In one embodiment, the muteins may e partially purified by ion exchange chromatography, and then further purified on a biotin-agarose column, for example. Muteins may be eluted off from such a column using biotin containing buffer as the eluant. Pure streptavidin mutein obtained by this simple procedure, after removal of biotin by dialysis, may then be used for biochemical characterizations.
Pure monomeric streptavidin muteins may be used to prepare affinity matrices for the purification of biotinylated proteins. A mutein of this invention can be coupled to a solid support using any one of a number of methods known to those skilled in the art, an example of which is disclosed in the Examples herein. “Solid support” as used herein includes any support that immobilizes the mutein of the invention, and includes specifically matrices or polymers such as agarose, polyacrylamide, cellulose, hydroxyapatite or dextran, surfaces of glass, plastic, silicone, ceramics or metals. The solid support may directly form a covalent bond with the mutein or it may be derivatized with a moiety that can form a covalent chemical bond with the mutein. An example of this type of covalent bond is the crosslinking via a carboxyl group, using crosslinking agents such as cyanogen bromide, glutaraldehyde, or hydroxysuccinimide. Alternatively, the solid support may be derivatized with a moiety that can form a near covalent bond with the mutein, for example an antibody to streptavidin.
Once immobilized to the solid support, the mutein will selectively bind biotin, for example in a mixture of biomolecules, and will release the bound biotin under relatively mild conditions, which do not destroy the biological activity of the biotin. In a preferred embodiment, the bound biotin is released by competition with another biotin moiety, however, other gentle methods including elution with salts, or by adjustment of pH, are intended to be included herein, and are known by those skilled in the art.
Biotinylated proteins may be purified by M4-agarose to high purity with a 75-80% recovery (FIG. 11). The operation is simpler than using monomeric avidin-agarose, which requires biotin pretreatment to saturate the non-exchangeable biotin binding sites in the matrix (Kohanski, 1990). Furthermore, recovery of some biotinylated proteins from the monomeric avidin-agarose is better achieved by elution with a solution of low pH. As well, regeneration of the matrix is usually achieved by washing at low pH. This exposure to extreme pH limits the potential application and durability of the monomeric avidin-agarose. The mild conditions in elution and regeneration with M4-agarose of the present invention are more likely to preserve biological activities of both the biotinylated proteins and the monomeric streptavidin in the matrix. One consequence of the mild conditions is that the M4-agarose matrix may be reused without any detectable loss in binding capacity.
EXAMPLES
The examples below are carried out using standard techniques, which are well known and routine to those skilled in the art. These examples are intended to be illustrative, but not limiting, of the invention.
Example 1
Construction of Streptavidin Mutants
Different point mutations were introduced to the coding sequence of a synthetic streptavidin gene (ssav) (SEQ ID NO:3) in the B. subtilis expression vector pSSAV-Tcry (Wu, 2002) by PCR-based oligonucleotide-directed mutagenesis. Five mutants (V125R, V125T, V55R, V55T and T76R), each bearing a single mutation which results in the change of an amino acid residue as the name suggests, were constructed using pSSAV-Tcry as the template and the primers listed in Table 3. The amplified products were digested with the pair of enzymes listed in Table 3 and cloned into pSSAV-Tcry. Five plasmids (pV125R, pV125T, pV55R, pV55T and pT76R) resulted.
Two double mutants (M2 and prior art AK) were also constructed. For M2 (T76R, V125R), a ScaI/NheI digested fragment of pV125R was used to replace the corresponding fragment in pT76R. For AK (D61A, W120K), the fragment bearing the two mutations was amplified by PCR using the primers SAVD61AF and SAVW120KB (Table 3) and the template pSSAV-Tcry. The amplified fragment was digested by XbaI/ScaI and used to replace the corresponding fragment in pSSAV-Tcry.
The construction of M4 (T76R, V125R, V55T, L109T) involved two steps (FIG. 1). First, a 164-bp fragment bearing two mutations (T76R, L109T) was amplified using SAVT76RF and SAVL109TB (Table 3) as primers and pSSAV-Tcry as template. The amplified product was digested by BamHI/ScaI and used to replace the corresponding fragment in pV55T to generate pV55T-T76R-L109T. In the second step, a ScaI/NheI digested fragment of pV125R was used to replace the corresponding fragment in pV55T-T76R-L109T to generate pV55T-T76R-L109T-V125R.








TABLE 3





Mutagenic primers for the construction of



streptavidin mutants. Mutated codons are


bolded and underlined. Restriction enzymes


in brackets behind the mutants refer to


the set of enzymes used for cloning.
















V125R, V125T (Scal/Sphl)










Forward primer:
SAVV125RTF




5′ GGAAAAGTACTCTTAC/GAGGACATGATAC



ATTTAC 3′



(SEQ ID NO: 4)


Backward primer:
pUB18H3



5′ GATTTCATACACGGTGCCTG 3′



(SEQ ID NO: 5)


 








V55R, V55T (Xbal/Sphl)










Forward primer:
SAVV55RTF




5′ GAATCTAGATACAC/GACTTACAGGAAGAT



ATG 3′



(SEQ ID NO: 6)


Backward primer:
pUB18H3


 








T76R (BamHl/Sphl)










Forward primer:
SAVT76RF




5′ GTGGATCCGGAACAGCACTTGGATGGAGAG



TT 3′



(SEQ ID NO: 7)


Backward primer: pUB18H3


 








D61A W120K (Xbal/Scal)










Forward primer:
SAVD61AF




5′ CATCTAGATACGTGCTTACAGGAAGATATG



CATCTGCACCT 3′



(SEQ ID NO: 8)


Backward primer:
SAVW120KB



5′ CAAGAGTACTTTTTTTTGCATTTGC



TTC 3′



(SEQ ID NO: 9)


 







T76R L109T (BamHl/Scal)









Forward primer:
SAVT76RF



Backward primer:
SAVL109TB



5′ GAGAGTACTTTTCCATGCATTTGCTTCTGT



TGTTCCAGATGTTAATGTCCATTGTGTG 3′



(SEQ ID NO: 10)









Example 2
Production and Purification of Streptavidin
Wild-type streptavidin was produced by B. subtilis WB800 [pSSAV-Tcry] cultured in a defined medium (Wu, 2002). The secreted protein was purified to homogeneity using cation exchange followed by iminobiotin affinity chromatography (Qureshi, 2001). Production and purification of streptavidin muteins followed a similar scheme with two major modifications: super-rich medium (Halling, 1977) was used in place of the defined medium and biotin-agarose (Sigma) was used in place of iminobiotin agarose as the affinity matrix. Dialyzed sample containing partially purified mutein was loaded to a 1-ml biotin-agarose column. Streptavidin muteins were eluted from the column using 20 mM d-biotin in phosphate-buffered saline (PBS; 50 mM sodium phosphate, 100 mM NaCl, pH 7.2). Concentration of purified streptavidin was determined spectrophotometrically using the known extinction coefficient at 280 nm (Gill, 1989; Gasteiger, 2003) for each individual mutein.
Example 3
Determination of the Molecular Size of Streptavidin
Molecular mass of purified streptavidin and its muteins was estimated by both gel filtration and dynamic light scattering studies. Gel filtration was performed on the Bio-Rad biologic workstation equipped with a Bio-Prep SE 100/17 column that had been calibrated with molecular mass protein markers (Bio-Rad). Molecular mass was also estimated from the hydrodynamic radius of the mutein obtained using a DynaPro MS dynamic light scattering instrument (Protein Solutions, USA) that had been calibrated with lysozyme. Protein samples (2-3 mg/ml in PBS) were passed through a 0.02 μm filter (Whatman Anodisc 13) immediately prior to measurement. The size distribution profile was analyzed using the manufacturer's Dynamics V6 software.
In single mutation muteins, the impact of the mutation on weakening of the subunit interaction followed the order T76R>V125R>V125T≈V55R>V55T (FIG. 3 and Table 4). The T76R mutein (referred to herein as M1) existed 100% in the monomeric state on the SDS-polyacrylamide gel even in the presence of biotin. In contrast, V55T mutation had the lowest impact, with the majority of molecules in the tetrameric state even in the absence of added biotin. Presence of biotin shifts the majority of the three remaining muteins (V125R, V125T and V55R) to the tetrameric state. Changing valine to arginine exerted greater impact than changing it to threonine. This is true for both Val-125 and Val-55.
Analysis of the solvent accessibility of individual amino acid residues with tetrameric streptavidin indicates that the solvent accessible area of Thr-76 in subunit A is zero. Its close distances to both Thr-76 and Arg-59 in subunit B and location on a rigid surface of a β-barrel structure make it an ideal residue to be changed to arginine to achieve the maximal electrostatic repulsion and steric hindrance effects at the subunit interface (FIG. 2B). The surface accessible area of Val-125 in subunit A is 1.78%. It has extensive interactions with Leu-109, Trp-120, Thr-123 and Val-125 in subunit D (FIG. 2C), Leu-109 in subunit B and Gln-107 in subunit C. Its conversion to arginine results in charge repulsion in subunit D and potential steric hindrance for subunits B, C, and D. As for Val-55 in subunit A, its surface accessible area is 29.7% and it is only close to Arg-59 in subunit B at the interface (FIG. 2D). Thus, V55R has the least impact on monomerization.







TABLE 4







Summary of the mutagenic effects on weakening of subunit


interactions in streptavidin muteins as reflected by the degree


of monomerization of streptavidin muteins in SDS-PAGE.


Estimation of the percentage of streptavidin in monomeric and


tetrameric states (number in the table) is based on the blots


showing migration pattern of non-boiled samples in FIG. 3 and 4.










No additional biotin
With additional biotin











Streptavidin mutein
Monomer
Tetramer
Monomer
Tetramer














M1 (T76R)
100
0
100
0


V125R
92
8
12
88


V125T
25
75
0
100


V55R
20
80
10
90


V55T
1
99
0
100


M2 (T76R, V125R)
100
0
100
0


M4 (T76R, V125R,
100
0
100
0


V55T, L109T)


AK
100
0
100
0









Observation of 100% monomerization of the streptavidin mutein using a non-boiled sample in SDS-PAGE does not always truly reflect its existence in the monomeric state in solution since SDS can promote subunit dissociation (Bayer, 1996; Waner, 2004). The apparent molecular masses of the purified wild-type streptavidin and the three muteins (M2, M4, AK) were estimated by gel filtration (FIG. 6A and Table 5). The expected molecular mass of monomeric streptavidin is 16.5 kDa. M2 and M4 in the absence of biotin showed the apparent molecular mass of 19.95 kDa and 21.87 kDa, respectively. These masses increased slightly in the presence of biotin. These data suggest that the muteins are monomeric in nature since their masses are less than that for the streptavidin dimer (33 kDa). In contrast, the AK mutein showed an apparent molecular mass of 45.66 kDa even in the absence of biotin. This indicates the oligomeric nature of this mutein. FIG. 6B shows the elution profile of purified M4 (in the absence of biotin) from the gel filtration column. The sample (loaded at 2 mg per ml) was eluted as a single peak. There is no evidence for the presence of tetrameric streptavidin, which would be eluted at 30.5 min.







TABLE 5







Molecular mass determination of wild-type (wt) streptavidin and its


muteins by gel filtration and dynamic light scattering. In dynamic


light scattering, the estimated molecular mass (M) was calculated


from the measured hydrodynamic radius (RH) using a


protein calibration curve. The peaks have a polydispersity below 15%.


Theoretical molecular mass is estimated from the amino acid


composition of the mature protein (Gasteiger, 2003).











RH (nm)
M (kDa)












M (kDa)
(Dynamic
M (kDa)


Sample
(Gel filtration)
light scattering)
(Theoretical)














Wt (no biotin)
56.23
3.69 ± 0.19
69
66.0






(tetramer)


AK (no biotin)
45.66
3.20 ± 0.23
50


AK (+biotin)
50.12
3.54 ± 0.38
63


M2 (no biotin)
19.95
1.98 ± 0.24
17
16.5






(monomer)


M2 (+biotin)
23.44
2.09 ± 0.29
18


M4 (no biotin)
21.87
2.08 ± 0.31
18.1
16.5






(monomer)


M4 (+biotin)
24.54
2.20 ± 0.14
21.5









Since the apparent molecular mass of wild-type streptavidin in the absence of biotin is 10 kDa less than expected (56 kDa instead of 66 kDa) as determined by gel filtration, dynamic light scattering (Schurr, 1977) was used as a second method to estimate the apparent molecular masses. The apparent molecular mass of wild-type streptavidin obtained in this way (69 kDa) was closer to that expected (66 kDa) (Table 5). The apparent molecular masses for both M2 and M4 in the absence of biotin indicated that they were in the monomeric state. Addition of biotin caused only a slight increase in their apparent molecular masses. The AK mutein again was found to be oligomeric independent of the presence or absence of biotin.
Example 4
Proteinase K Digestion of Streptavidin and its Muteins
Purified streptavidin and its muteins (30 μM monomer) were treated with proteinase K (Invitrogen, 5 μM) for 15 min at 30° C. in 50 mM Tris-HCl containing 5 mM CaCl2, pH 8.0. The reaction was stopped by precipitation with trichloroacetic acid (Ellison, 1995). Boiled samples of precipitated proteins were resolved by reducing SDS-PAGE. The same analysis was performed with streptavidin samples treated with biotin (1 mM final concentration) prior to proteinase K digestion.
Monomeric streptavidin is expected to be more susceptible to proteinase K digestion (Laitinen, 2003). Therefore, wild type streptavidin and its muteins were treated with proteinase K (FIG. 7A). Wild type streptavidin was converted to the core form independent of the presence or absence of biotin. Under the condition used, the core streptavidin was resistant to further degradation by proteinase K. In contrast, all three muteins including AK, M2 and M4 were much more susceptible to proteinase K digestion. Sensitivity to proteinase K is more apparent for M2 and M4, which were completely digested independent of the presence or absence of biotin. This property is consistent with the monomeric nature of these muteins. The AK mutein behaved differently. While most of it was digested by proteinase K in the absence of biotin, it became much more resistant to proteinase K when biotin was present.
Example 5
Cross-Linking Reactions
Cross-linking of streptavidin and its muteins was carried out using sulfo-EGS [ethylene glycolbis(sulfosuccinimidyl succinate)] (Pierce) as the cross-linker. A typical reaction mixture (20 μl) contained the purified mutein (0.25 mg/ml) and sulfo-EGS (10-fold molar excess over the protein) in PBS. After 30 min at room temperature, the reaction was quenched with Tris-HCl (30 mM, pH 7.5). Aliquots of the cross-linking reaction samples were boiled and examined by SDS-PAGE. Lysozyme (Sigma, 0.25 mg/ml) was included in the study to help establish the optimal reaction conditions.
To strengthen the idea that both M2 and M4 are monomeric while AK is oligomeric in nature, protein cross-linking was carried out using sulfo-EGS as the cross-linking agent. Sulfo-EGS reacts with both the accessible α-amino groups at the N-termini and the surface exposed ε-amino groups of the lysine side-chains in proteins. Secreted wild type streptavidin has eight lysine residues in each subunit. Three dimensional structural model of streptavidin suggests that lysine-121 in subunit A is 14.1 Å from lysine-121 in subunit D. As the spacer arm in sulfo-EGS is 16.1 Å, subunits A and D (same for subunits B and C) should be easily cross-linked by sulfo-EGS. Also, it is possible to have cross-linking between subunits A and B as the N-terminal region from subunit A which contains two lysine residues is likely to be positioned close to lysine-80 in subunit B. The same is true for subunits C and D. Therefore, one should be able to differentiate tetrameric streptavidin from the monomeric form with the observation of cross-linked tetrameric streptavidin using sulfo-EGS. Lysozyme, well known to be monomeric in solution (Blake, 1965; Schwalbe, 2001), served as the negative control. FIG. 7B shows that the amount of dimeric lysozyme increased slightly in the presence of the cross-linking agent. This helped set the upper limit of the concentration of sulfo-EGS to be used under the experimental condition. The wild type streptavidin subunit had an apparent molecular mass of 19 kDa on the SDS-gel. After treatment with sulfo-EGS, most of these subunits were cross-linked to dimers and higher oligomers with small amounts remaining in the monomeric state. M2 and M4 muteins behaved very similarly (data for M2 are not shown). The majority of the M2 and M4 muteins after the cross-linking treatment migrated as monomers with small amounts in the dimeric form. These dimers may represent cross-linked monomeric subunits, which were artificially generated in the same manner as with lysozyme. AK showed a cross-linking profile very similar to that of the wild type streptavidin. These data strongly support the idea that M2 and M4 muteins are monomeric while the AK mutein is oligomeric in solution, respectively.
Example 6
Kinetic Analysis of Streptavidin Muteins
The kinetic parameters (both on- and off-rates for interaction with biotin) of streptavidin muteins were determined in real time using the surface plasmon resonance based BIAcoreX biosensor. Biotin-conjugated bovine serum albumin immobilized on CM5 sensor chip was used to study the reversibility of biotin binding (Qureshi, 2001).
As shown in Table 6 (graphical plots for M4 are shown in FIG. 8), M2, M4 and AK had their dissociation constant (Kd) in the range of 10−7M. The off-rates (kd) for these muteins were almost the same while the on-rate (ka) for the AK mutein was slightly lower than the rest. One of the factors affecting on-rate is the diffusion coefficient (or molecular mass) of the streptavidin molecule. Since AK is oligomeric in nature, this may account for the lower on-rate for this mutein-biotin interaction.







TABLE 6







Kinetic parameters for the interactions between


streptavidin muteins and biotin.










Protein
ka (M−1s−1)
kd (s−1)
Kd (M)





M2
1.88 ± 0.07 × 104
3.22 ± 0.01 × 10−3
1.71 ± 0.07 × 10−7


M4
1.80 ± 0.04 × 104
3.39 ± 0.02 × 10−3
1.87 ± 0.06 × 10−7


AK
1.46 ± 0.05 × 104
3.59 ± 0.03 × 10−3
2.46 ± 0.10 × 10−7





ka, on-rate;


kd, off-rate;


Kd = kd/ka.






Example 7
Computer Programs for Analyses of Streptavidin
Swiss-pdb Viewer (Guex, 1997) was used to display streptavidin (1SWE (20)), analyze interfacial residues, measure distance between residues and align the structures of streptavidin and avidin. Interfacial contact areas were calculated using the protein-protein interaction server (Jones, 1995) and the Formiga module in the Sting Millennium Suite (Neshich, 2003). The plots of accessible surface area of individual residues in streptavidin in either the monomeric or tetrameric state were generated using the Protein Dossier module in the Sting Millennium Suite.
Example 8
Construction of pET-SAVM4
Plasmid pET-SAVM4 is an expression vector for producing the monomeric streptavidin mutein M4 from E. coli using the T7 promoter system. The streptavidin gene bearing 4 mutations was amplified by PCR with the B. subtilis plasmid p(T76R, V125R, V55T, L109T) (Bashir, 2001) as the template and synthetic oligonucleotides ECSAVF (5′TGGACCCGAGCAAAGATTCTAAAG 3′ SEQ ID NO:10) and ECSAVB (5′ CTGTCGACTGACTGCGTTAGCAATTTAAC 3′ SEQ ID NO:11) as the forward and backward primers, respectively. The 714-bp amplified product was phosphorylated by T4 polynucleotide kinase and digested with SalI. It was then inserted in-frame to pET29b (Novagen) which has been digested with NdeI (followed by a fill-in reaction to generate a blunt end) and SalI. The resulting plasmid, designated pET-SAVM4, was transformed to E. coli BL21(DE3) (Novagen) for expression studies.
Example 9
Cell Growth
E. coli BL21(DE3)[pET-SAVM4] was grown at 37° C. in Luria broth (1% tryptone, 0.5% yeast extract, 1% NaCl) containing 30 μg/ml kanamycin to 150-200 Klett units in a shake flask. IPTG was then added to a final concentration of 0.1 mM and growth continued for 4-5 hours at 26-28° C. Cell density was measured using a Klett-Summerson photoelectric colorimeter with a green filter (Klett Mfg. Co; New York).
Example 10
Purification of Monomeric Streptavidin
Cells of E. coli BL21(DE3)[pET-SAVM4] were harvested by centrifugation at 12,000 g for 5 min at 4° C. The cell pellet was resuspended in buffer (30 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and disrupted with a French press. The crude cell lysate was separated into soluble and insoluble fractions by centrifugation at 20,000 g for 20 min. The soluble fraction was dialysed overnight against phosphate-buffered saline (PBS; 50 mM sodium phosphate, 100 mM NaCl, pH 7.2) to remove any biotin contaminants. Monomeric streptavidin in the dialysate was purified on a biotin-agarose column (Sigma) (Wu, 2005). Concentration of purified streptavidin was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 41,820 M−1cm−1 for calculation (Gill, 1989).
Example 11
Preparation of Immobilized Monomeric Streptavidin
Pure monomeric M4 streptavidin mutein was used to prepare affinity matrix for the purification of biotinylated proteins. The mutein was coupled to cyanogen-bromide activated agarose (Sigma) according to the manufacturer's instructions. The amount of non-immobilized streptavidin was estimated by colorimetric method using a protein assay reagent (Bio-Rad). The coupling efficiency was determined to be around 70%.
Example 12
Affinity Purification of Biotinylated Proteins
The purification of two biotinylated proteins from a crude sample was used to demonstrate the functionality of the M4 affinity matrix. One protein was chemically biotinylated horse heart cytochrome c (5.5 moles biotin/mole cytochrome c, Sigma); the other was enzymatically biotinylated maltose binding protein-AviTag fusion (MBP-AviTag; 1 mole biotin/mol protein, Avidity). Each protein was mixed with a crude B. subtilis cellular extract that had been pretreated to remove endogenous biotin and biotinylated proteins (Qureshi, 2002). The sample was applied to a 200:1-column of M4 affinity matrix which had been equilibrated in PBS. After washing the matrix with 4-5 column volumes of PBS, the bound proteins were eluted with 10 mM biotin in PBS. The fractions collected were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and, for biotinylated cytochrome c, also by spectrophotometric measurement at 550 nm for the ∀-peak of the reduced heme c moiety of cytochrome c. Regeneration of M4 column and determination of the full binding capacity of the affinity matrix followed the method in an earlier study (Qureshi, 2002).
Example 13
Other Methods
Vent DNA polymerase (New England BioLabs) was used for DNA amplification. The sequence of the PCR product was confirmed to be free of amplification errors by nucleotide sequencing performed at the University Core DNA and protein services, University of Calgary, Calgary, Alberta, Canada. Molecular mass of M4 was determined by SELDI-TOF mass spectrometry on an NP20 chip using sinapic acid as the matrix. The chip array was analyzed in a PBS (Protein Biosystem) II ProteinChip Reader (Ciphergen Biosystems) as described in a previous article (Wu, 2005). The kinetic parameters for monomeric streptavidin to interact with biotinylated bovine serum albumin (BSA) were determined using BIAcore X biosensor as previously described (Qureshi, 2002).
REFERENCES
The following references are referred in parenthesis in the above description and are incorporated herein as if reproduced in their entirety.

    Atwell, S., Ultsch, M., De Vos, A. M., and Wells, J. A. (1997) Science 278, 1125-1128
    Bashir, R., Gomez, R., Sarikaya, A., Ladisch, M. R., Sturgis, J., and Robinson, J. P. (2001) Adsorption of avidin on microfabricated surfaces for protein biochip applications. Biotechnol. Bioeng. 73, 324-328.
    Bayer, E. A., and Wilchek, M. (1990) Application of avidin-biotin technology to affinity based separations. J. Chromatography. 510, 3-11.
    Bayer, E. A., Ehrlichrogozinski, S., and Wilchek, M., (1996) Sodium dodecyl sulfate polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin. Electrophoresis. 17, 1319-1324.
    Bayer, E. A., Ben Hur, H., and Wilchek, M., Isolation and properties of streptavidin, Methods Enzymol. 184:80-9 (1990) 80-89.
    Blake, C. C., Koenig, D. F., Mair, G. A., North, A. C., Phillips, D. C., and Sarma, V. R. (1965) Nature 206, 757-761
    Casalini, P., Luison, E., Menard, S., Colnaghi, M. I., Paganelli, G., and Canevari, S. (1997) Tumor pretargeting: role of avidin/streptavidin on monoclonal antibody internalisation. J. Nucl. Med. 38, 1378-1381.
    Dieckman, L., Gu, M., Stols, L., Donnelly, M. I., and Collart, F. R. (2002) High throughput methods for gene cloning and expression. Protein Expr. Purif. 25, 1-7.
    Ellison, D., Hinton, J., Hubbard, S. J., and Beynon, R. J. (1995) Protein Sci. 4, 1337-1345
    Freitag, S., Le Trong, I., Chilkoti, A., Klumb, L. A., Stayton, P. S., and Stankamp, R. E. (1998) Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex. J. Mol. Biol. 279, 211-221.
    Freitag, S., Le Trong, I., Chilkoti, A., Klumb, L. A., Stayton, P. S., and Stenkamp, R. E., Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex, J. Mol. Biol. 279 (1998) 211-221.
    Gallizia, A., de Lalla, C., Nardone, E., Santambrogio, P., Brandazza, A., Sidoli, A., and Arosio, P., Production of a soluble and functional recombinant streptavidin in Escherichia coli, Protein Expr. Purif. 14 (1998) 192-196.
    Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., and Bairoch, A. (2003) Nucleic Acids Res. 31, 3784-3788
    Gill, S. C. and Von Hippel, P. H., (1989) Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem. 182, 319-326.
    Gonzalez, M., Argarana, C. E., and Fidelio, G. D. (1999) Biomol. Eng 16, 67-72
    Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184, 51-67.
    Guex, N. and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723
    Halling, S. M., Sanchez-Anzaldo, F. J., Fukuda, R., Doi, R. H., and Meares, C. F. (1977) Biochemistry 16, 2880-2884
    Hendrickson, W. A., Pahler, A., Smith, J. L., Satow, Y., Merritt, E. A., and Phizackerley, R. P., (1989) Crystal structure of core streptavidin determined from multi-wavelength anomalous diffraction of synchrotron radiation. Proc. Natl. Acad. Sci. U.S.A. 86, 2190-2194.
    Henrikson, K. P., Allen, S. H., and Maloy, W. L., (1979) An avidin monomer affinity column for the purification of biotin-containing enzymes. Anal. Biochem. 94, 366-370.
    Hunt, I., (2005) From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expr. Purif. 40, 1-22.
    Hyre, D. E., Le, Trong, I, Freitag, S., Stenkamp, R. E., and Stayton, P. S., Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system, Protein Sci. 9 (2000) 878-885.
    Jones, S, and Thornton, J. M. (1995) Protein-protein interactions: a review of protein dimer structures. Prog. Biophys. Mol. Biol. 63, 31-65.
    Kohanski, R. A., and Lane, M. D., (1990) Monovalent avidin affinity columns. Methods Enzymol. 184, 194-200.
    Laitinen, O. H., Marttila, A. T., Airenne, K. J., Kulik, T., Livnah, O., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (2001) Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions. J. Biol. Chem. 276, 8219-8224.
    Laitinen, O. H., Nordlund, H. R., Hytonen, V. P., Uotila, S. T., Marttila, A. T., Savolainen, J., Airenne, K. J., Livnah, O., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (2003) Rational design of an active avidin monomer. J. Biol. Chem. 278, 4010-4014.
    Lesley, S. A. (2001) High-throughput proteomics: protein expression and purification in the postgenomic world. Protein Expr. Purif. 22, 159-164.
    Lichty, J. J., Malecki, J. L., Angew, H. D., Michelson-Horowitz, D. J., and Tan, S., (2005) Comparison of affinity tag for protein purification. Protein Expr. Purif., 41, 98-105.
    Liu, Q., Li, M. Z., Leibham, D., Cortez, D., and Elledge, S. J. (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300-1309.
    Livnah, O., Bayer, E. A., Wilchek, M., and Sussman, J. L. (1993) Three-dimensional structures of avidin and avidin-biotin complex. Proc. Natl. Acad. Sci. U.S.A. 90, 5076-5080.
    Marttila, A. T., Airenne, K. J., Laitinen, O. H., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (1998) FEBS Lett. 441, 313-317
    Marttila, A. T., Laitinen, O. H., Airenne, K. J., Kulik, T., Bayer, E. A., Wilchek, M., and Kulomaa, M. S. (2000) FEBS Lett. 467, 31-36
    Neshich, G., Togawa, R. C., Mancini, A. L., Kuser, P. R., Yamagishi, M. E., Pappas, G., Jr., Torres, W. V., Fonseca e Campos, Ferreira, L. L., Luna, F. M., Oliveira, A. G., Miura, R. T., Inoue, M. K., Horita, L. G., de Souza, D. F., Dominiquini, F., Alvaro, A., Lima, C. S., Ogawa, F. O., Gomes, G. B., Palandrani, J. F., dos Santos, G. F., de Freitas, E. M., Mattiuz, A. R., Costa, I. C., de Almeida, C. L., Souza, S., Baudet, C., and Higa, R. H. (2003) Nucleic Acids Res. 31, 3386-3392
    Niemeyer, C. M., Adler, M., Pignataro, B., Lenhert, S., Gao, S., Chi, L., Fuchs, H., and Blohm, D. (1999) Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR. Nucleic Acids Res. 27, 4553-3451.
    Pahler, A., Hendrickson, W. A., Kolks, M. A., Argarana, C. E., and Cantor, C. R., Characterization and crystallization of core streptavidin, J. Biol. Chem. 262 (1987) 13933-13937.
    Qureshi, M. H., Yeung, J. C., Wu, S.-C. and Wong, S.-L. (2001) Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities. J. Biol. Chem. 276, 46422-46428.
    Qureshi, M. H. and Wong, S.-L., (2002) Design, production and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins. Protein Expr. Purif. 25, 409-415.
    Sano, T. and Cantor, C. R. (1995) Intersubunit contacts made by trytophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc. Natl. Acad. Sci. U.S.A. 92, 3180-3184.
    Sano, T., Pandori, M. W., Chen, X. M., Smith, C. L., Cantor, C. R., and Chen, X., Recombinant core streptavidins—A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules, J. Biol. Chem. 270 (1995) 28204-28209.
    Schechter, B., Chen, L., Amon, R., and Wilchek, M., (1999) Organ selective delivery using a tissue-directed streptavidin-biotin system: targeting 5-fluorouridine via TNP-streptavidin. J. Drug Target. 6, 337-348.
    Scholle, M. D., Collart, F. R., and Kay, B. K. (2004) In vivo biotinylated proteins as targets for phage-display selection experiments. Protein Expr. Purif. 37, 243-252.
    Schurr, J. M. (1977) CRC Crit. Rev. Biochem. 4, 371-431
    Schwalbe, H., Grimshaw, S. B., Spencer, A., Buck, M., Boyd, J., Dobson, C. M., Redfield, C., and Smith, L. J. (2001) Protein Sci. 10, 677-688
    Sorensen, H. P., Sperling-Petersen, H. U., and Mortensen, K. K., A favorable solubility partner for the recombinant expression of streptavidin, Protein Expr. Purif. 32 (2003) 252-259.
    Swint-Kruse, L., Elam, C. R., Lin, J. W., Wycuff, D. R., and Shive, M. K. (2001) Protein Sci. 10, 262-276
    Szafranski, P., Mello, C. M., Sano, T., Smith, C. L., Kaplan, D. L., and Cantor, C. R., A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system, Proc. Natl. Acad. Sci. U.S. A 94 (1997) 1059-1063.
    Terpe, K., (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533.
    Vetter, I. R., Baase, W. A., Heinz, D. W., Xiong, J. P., Snow, S., and Matthews, B. W. (1996) Protein Sci. 5, 2399-2415
    Waner, M. J., Navrotskaya, I., Bain, A., Oldham, E. D., and Mascotti, D. P., (2004) Thermal and sodium dodecylsulfate induced transitions of streptavidin. Biophys. J. 87, 2701-2713.
    Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. and Salemme, F. R. (1989) Structural origins of high-affinity biotin binding to streptavidin. Science. 243, 85-88.
    Wilchek, M. and Bayer, E. A. (1988) The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171, 1-32.
    Wilchek, M. and Bayer, E. A. (1990) Introduction to avidin-biotin technology. Methods Enzyymol. 184, 5-13.
    Wong, S.-L., X.-C. Wu, L.-P. Yang, S.-C. Ng, and N. Hudson. 1995. Production and purification of antibody using Bacillus subtilis as an expression host, p. 100-107. In H. Y. Wang and T. Imanaka (eds.), Antibody Expression and Engineering. American Chemical Society, Washington, D.C.)
    Wu, S.-C. and Wong, S.-L. (2005) Engineering soluble monomeric streptavidin with reversible biotin binding capability. J. Biol. Chem. 280, 23225-23231.
    Wu, S.-C., Qureshi, M. H., and Wong, S.-L. (2002) Protein Expr. Purif: 24, 348-356

## Claims

1. A method of isolating a biotinylated molecule from a sample comprising the steps of binding a streptavidin mutein to a solid support to form a matrix, passing the sample over the support, and eluting the biotinylated molecule, wherein the streptavidin mutein comprises an amino acid sequence having Thr76 of SEQ ID NO:1 substituted with a charged amino acid.

2. The method of claim 1, comprising the further step of regenerating the matrix for reuse.

3. The method of claim 1, wherein said mutein further comprises at least one mutation of a hydrophobic interfacial amino acid residue to a less hydrophobic, charged or hydrophilic residue.

4. The method of claim 3, wherein at least one mutation is selected from the group consisting of:
a) Val125 of SEQ ID NO:1 substituted with a charged or hydrophilic amino acid;
b) Val55 of SEQ ID NO:1 substituted with a charged or hydrophilic amino acid; and
c) Leu109 of SEQ ID NO:1 substituted with a charged or hydrophilic amino acid.

5. The method of claim 1, wherein Thr76 of SEQ ID NO:1 is substituted with Arg, Lys or His.

6. The method of claim 5, wherein Thr76 of SEQ ID NO:1 is substituted with Arg.

7. The method of claim 5, wherein Thr76 of SEQ ID NO:1 is substituted with Lys.

8. The method of claim 5, wherein Thr76 of SEQ ID NO:1 is substituted with His.

9. The method of claim 4, wherein Val125 of SEQ ID NO:1 is substituted with Arg, Lys or His.

10. The method of claim 9, wherein Val125 of SEQ ID NO:1 is substituted with Arg.

11. The method of claim 4, wherein Val55 of SEQ ID NO:1 is substituted with Thr or Ser.

12. The method of claim 11, wherein Val55 of SEQ ID NO:1 is substituted with Thr.

13. The method of claim 4, wherein Leu109 of SEQ ID NO: 1 is substituted with Thr or Ser.

14. The method of claim 13, wherein Leu109 of SEQ ID NO:1 is substituted with Thr.

15. The method of claim 4, wherein said mutein further comprises at least four mutations where Thr76 of SEQ ID NO:1 is substituted with Arg, Val125 is substituted with Arg, Val55 is substituted with Thr; and Leu109 is substituted with Thr.

16. The method of claim 1, wherein said mutein exists in monomeric form in solution in the presence of biotin.

17. The method of claim 1, wherein the mutation is chosen to cause one or more of steric hindrance, charge repulsion, and improvement of solubility by changing interfacial hydrophobic residues to less hydrophobic or hydrophilic ones.