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PubMed1990

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro.

We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.

Adenosine Triphosphatases↗

PubMed2005

Genetic deletion of murine SPRY domain-containing SOCS box protein 2 (SSB-2) results in very mild thrombocytopenia.

The SSB family is comprised of four highly homologous proteins containing a C-terminal SOCS box motif and a central SPRY domain. No function has yet been ascribed to any member of this family in mammalian species despite a clear role for other SOCS proteins in negative regulation of cytokine signaling. To investigate its physiological role, the murine Ssb-2 gene was deleted by homologous recombination. SSB-2-deficient mice were shown to have a reduced rate of platelet production, resulting in very mild thrombocytopenia (25% decrease in circulating platelets). Tissue histology and other hematological parameters were normal, as was the majority of serum biochemistry, with the exception that blood urea nitrogen (BUN) levels were decreased in mice lacking SSB-2. Quantitative analysis of SSB mRNA levels indicated that SSB-1, -2, and -3 were ubiquitously expressed; however, SSB-4 was only expressed at very low levels. SSB-2 expression was observed in the kidney and in megakaryocytes, a finding consistent with the phenotype of mice lacking this gene. Deletion of SSB-2 thus perturbs the steady-state level of two tightly controlled homeostatic parameters and identifies a critical role for SSB-2 in regulating platelet production and BUN levels.

Amino Acid Motifs↗

PubMed1994

Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions.

We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB)65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering approximately 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to dimers of tetramers (octamers). The intrinsic association equilibrium constant, K(obs), and the "limited" cooperativity parameter, omega T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, omega T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K(obs) decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K(obs) on [NaF] is independent of pH with delta logK(obs)/delta log[NaF] = -4.5(+/- 0.5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (delta logK(obs)/delta log[NaCl]) ranges from -12.0(+/- 0.8) at pH 5.5, to -6.0(+/- 0.5) at pH 9.0 (at 25 degrees C). Thus Cl- release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F-. The linkages of ion concentration and pH on K(obs) can be described in terms of: (1) cation release from the polynucleotide; (2) release of Cl- from sites on the SSB tetramer that require protonation to bind Cl-; (3) binding of cations to sites on the SSB tetramer which require deprotonation for cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.

Anions↗

PubMed1998

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex.

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Adenosine Triphosphate↗

PubMed1992

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46.

Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1. A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113. It has very little or no effect on ssb-1 or ssb-3. All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels. The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda. The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411. The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain.

Bacterial Proteins↗

PubMed1983

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli.

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene; the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage lambda +, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA- mutant strains and will provide a means of transferring ssb- mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.

Bacterial Proteins↗

PubMed2006

Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA.

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (DeltaH(obs)) and the negative heat capacity change (DeltaC(p,obs)) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined DeltaH(obs) over a much wider temperature range (5-60 degrees C) and as a function of monovalent salt concentration and type for SSB binding to (dT)(70) under solution conditions that favor the fully wrapped (SSB)(65) complex (monovalent salt concentration >or=0.20 M). Over this wider temperature range we observe a strongly temperature-dependent DeltaC(p,obs). The DeltaH(obs) decreases as temperature increases from 5 to 35 degrees C (DeltaC(p,obs) <0) but then increases at higher temperatures up to 60 degrees C (DeltaC(p,obs) >0). Both salt concentration and anion type have large effects on DeltaH(obs) and DeltaC(p,obs). These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 degrees C), the midpoint of which shifts to higher temperature (above 35 degrees C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br(-) > Cl(-) > F(-), and these anions are then released upon binding ssDNA, affecting both DeltaH(obs) and DeltaC(p,obs). We conclude that the experimentally measured values of DeltaC(p,obs) for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.

Anions↗

PubMed2005

The SPRY domain of SSB-2 adopts a novel fold that presents conserved Par-4-binding residues.

The four mammalian SPRY domain-containing SOCS box proteins (SSB-1 to SSB-4) are characterized by a C-terminal SOCS box and a central SPRY domain. We have determined the first SPRY-domain structure, as part of SSB-2, by NMR. This domain adopts a novel fold consisting of a beta-sandwich structure formed by two four-stranded antiparallel beta-sheets with a unique topology. We demonstrate that SSB-1, SSB-2 and SSB-4, but not SSB-3, bind prostate apoptosis response protein-4 (Par-4). Mutational analysis of SSB-2 loop regions identified conserved structural determinants for its interaction with Par-4 and the hepatocyte growth factor receptor, c-Met. Mutations in analogous loop regions of pyrin and midline-1 SPRY domains have been shown to cause Mediterranean fever and Opitz syndrome, respectively. Our findings provide a template for SPRY-domain structure and an insight into the mechanism of SPRY-protein interaction.

Amino Acid Motifs↗

PubMed1990

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs.

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single-strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein.

Antibodies↗

PubMed2000

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins.

DNA replication of phi29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of phi29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of phi29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during phi29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by phi29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the phi29 and Nf SSBs do not show any effect. On the other hand, the phi29 SSB is the only one of the three SSBs able to increase the replication rate of phi29 DNA polymerase in primed M13 ssDNA replication. From the fact that the phi29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of phi29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on phi29-like DNA replication has been proposed.

Amino Acid Sequence↗

PubMed2004

PriA helicase and SSB interact physically and functionally.

PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination. We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB. This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB. Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops. Correlation between the DNA repair and recombination defects of strains harbouring an SSB C-terminal mutation with inhibition of this SSB-PriA interaction in vitro suggests that SSB plays a critical role in facilitating PriA-directed replication restart. Taken together with previous data, these findings indicate that protein-protein interactions involving SSB may coordinate replication fork reloading from start to finish.

Adenosine Triphosphatases↗

PubMed1999

SSB, encoding a ribosome-associated chaperone, is coordinately regulated with ribosomal protein genes.

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

Alleles↗

PubMed1999

Detecting anti-SSA and anti-SSB antibodies in routine analysis: a comparison between double immunodiffusion and immunoblotting.

The aim of this study was to assess the performance of a commercially available procedure for detecting anti-Sjögren's syndrome A (anti-SSA) and anti-Sjögren's syndrome B (anti-SSB) antibodies by immunoblotting (IB) and compare it with double immunodiffusion (DID). We also studied the clinical significance of these profiles in a series of unselected anti-SSA positive patients. Serum samples from 534 patients that were positive on an immunofluorescent screening test using HEp-2 cells were analysed for anti-SSA and anti-SSB antibodies by DID and IB (Biolab Anablot System II), and the results on anti-SSA antibodies were confirmed by an enzyme-linked immunosorbent assay (ELISA). Fifty-five serum samples were found to be positive for anti-SSA antibodies. Among these, 24 were anti-SSA negative by IB but positive by DID and ELISA ('non-blotter sera'), whereas only three serum samples were anti-SSA negative by DID but positive by IB and ELISA. Of the 18 anti-SSB positive serum samples, eight were negative by DID. All the serum samples that were anti-SSB positive by DID were also positive by IB. Anti-SSB antibodies showed a significant association with eye dryness and leucopenia. Anti-52 kDa SSA antibodies were associated with anti-SSB antibodies but showed no significant association with sicca symptoms, while anti-60 kDa SSA antibodies were associated with lower rates of leucopenia. The 'non-blotter' profile showed no significant association with any clinical parameter. IB is less sensitive than DID for detecting anti-SSA antibodies but more sensitive than DID for detecting anti-SSB antibodies. The determination of anti-SSA immunoblotting profiles in patients positive for anti-SSA antibodies by DID does not significantly improve the clinical usefulness of this test. As expected, anti-SSB antibodies were associated with clinical features of Sjögren's disease. Non-blotting (probably conformational) anti-SSA antibodies did not show any further association with clinical parameters and seem to have no clinical relevance.

Autoantibodies↗

PubMedDate not supplied

In vitro autoantibody synthesis by anti-La(SSB) positive SLE patients and healthy controls.

In vitro production of anti-La(SSB) and anti-DNA antibodies by mononuclear cells (mnc) from systemic lupus erythematosus (SLE) patients and healthy controls was measured using ELISA techniques. The SLE patients were divided into anti-La(SSB) positive and negative groups depending on Ouchterlony analysis of the serum. Both patient groups spontaneously produced raised levels of total IgG but not IgM compared to controls. Pokeweed mitogen (PWM) stimulated mnc from anti-La(SSB) positive SLE patients and controls gave good total IgM and IgG responses. In contrast the anti-La(SSB) negative group gave no IgG but did make an IgM response. Kinetic studies showed that 14 day cultures were essential to produce detectable amounts of specific autoantibodies which, with a few exceptions, were of IgM isotype. Low levels of anti-La(SSB) and anti-DNA were spontaneously produced by anti-La(SSB) positive SLE patients and controls. PWM stimulation at least doubled autoantibody synthesis in these two groups. The anti-La(SSB) negative SLE group gave similar responses although PWM induced autoantibody levels were reduced. ConA-induced suppressor cell assay showed that anti-La(SSB) positive SLE patients could form suppressor cells but not as effectively as controls. This group of SLE patients were more successful at suppressing IgG than IgM responses.

Adult↗

PubMed1993

Detection of anti-SSB antibodies in patients with rheumatic diseases.

Anti-SSB antibodies were measured by ELISA in patients with various kinds of connective tissue diseases using SSB antigen purified from fresh rabbit thymus. The SSB antigen reacted with anti-SSB standard serum, and the positive rates in SS, SLE, RA, PBS and MCTD were 55.1%, 48.3%, 32.8%, 30.8% and 26.3%, respectively. The titers of anti-SSB antibodies were higher in SS and SLE patients than in other connective tissue disease patients. However, 10% of normal individuals were found to have anti-SSB antibodies with low titers. The anti-SSB antibodies detected were mainly of IgG isotype. Preliminary analysis of clinical data showed no relationship between anti-SSB and systemic involvement in SS.

Antibodies, Antinuclear↗

PubMed1996

Subclass distribution of maternal and neonatal anti-Ro(SSA) and La(SSB) antibodies in congenital heart block.

OBJECTIVE: To compare the subclass distribution of anti-48 kDa La(SSB) and anti-52 and 60 kDa Ro(SSA) antibodies in the maternal and neonatal circulation, in pregnancies affected and unaffected by the development of congenital heart block (CHB). METHODS: Sera were obtained from 32 mothers (during 34 pregnancies 23 complicated by CHB and 11 healthy) demonstrated to have anti-Ro(SSA) and/or La(SSB). Maternal and neonatal autoantibodies were evaluated for subclass distribution by ELISA: RESULTS: All 4 subclasses of anti-Ro(SSA) and La(SSB) antibodies cross the placenta and are detectable in sera obtained from the umbilical cord, IgG1 and IgG3 were the major subclasses represented in the 48 kDa La(SSB) and 52 kDa Ro(SSA) responses. All subclasses, including IgG2 and IgG4, were observed in about one-third of the anti-52 kDa Ro(SSA) and 48 kDa La(SSB) responses. In contrast, anti-60 kDa antibodies were, with rare exception, confined to IgG1. Except for anti-48 kDa La(SSB) IgG3 antibodies, no significant differences were observed between affected and unaffected pregnancies in the ratio of maternal to neonatal levels of any of the antibody subclasses. Overall, there were no significant differences in the subclass profiles between mothers whose children had heart block and those who did not. CONCLUSION: The IgG subclasses of anti-48 kDa La(SSB) and anti-52 and 60 kDa Ro(SSA) do not account for the susceptibility of one fetus versus another for the development of CHB. Anti-60 kDa Ro(SSA) antibodies are more restricted in subclass distribution than anti-52 kDA Ro(SSA) or 48 kDa La(SSB) responses.

Antibodies, Antinuclear↗

PubMed1997

T cell reactivity to Sjögren's syndrome related antigen La(SSB).

OBJECTIVE: Many patients with primary Sjögren's syndrome (SS) make high titer IgG autoantibodies to the La(SSB) antigen, suggesting antigen specific T cell-B cell interactions. T cell responses to some nuclear antigens, particularly U1RNP, have been detected in patients with systemic lupus erythematosus (SLE) and in healthy subjects. We investigated T cell reactivity to the autoantigen SSB in patients with SS and healthy controls. METHODS: Using the [3H]thymidine proliferation assay, we determined reactivity to purified recombinant SSB (rSSB) in 20 patients with SS and 19 controls. Specificity was determined using tetanus toxoid, endotoxin, and 3 other autoantigens (PBC.M2, Sc170, and GAD). Precursor frequency was calculated by limiting dilution analysis. HLA Class II dependency was investigated using anti-Class II monoclonal antibodies. HLA-DR typing was by polymerase chain reaction and sequence specific oligonucleotide typing. RESULTS: Six of 20 patients with SS and 10/19 controls proliferated to La(rSSB). Precursor frequency of anti-SSB T cells was 1:77,040 and 1:115,000 in 2 healthy subjects and 1:230,250 and 1:103,034 in two patients with SS. Anti-HLA-DR abrogated proliferation to SSB and tetanus toxoid. Thirteen of 15 patients with SS and 4/17 controls were HLA-DR3 positive, with no apparent association of HLA-DR3 with SSB reactivity in controls. CONCLUSION: Anti-La(SSB) specific T cells occur in a significant proportion of controls and in some patients with SS. The function of SSB T cells in controls remains to be defined. They may represent immunoregulatory cells, and further analysis of these cells, and a comparison to those found in patients with SS, may elucidate normal immunoregulation and the derangements that lead to Sjögren's syndrome.

Adult↗

PubMed1999

Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification.

Polymerase chain reaction (PCR)-derived DNA fragment containing the complete structural gene for SSB protein of the Escherichia coli was cloned into an expression vector. The clones expressing His-tagged SSB protein were selected. The cloned DNA fragments were verified to be authentic by sequencing several clones. The recombinant SSB protein (His-tagged SSB) contained a polyhistidine tag at the N-terminus (38 additional amino acids) that allowed single-step isolation by Ni2+ affinity chromatography. We found that recombinant plasmids are unstable and give a low level of expression in E. coli BL21(DE3) strain. However, the plasmids were stable in E. coli BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were highly expressed upon IPTG addition. The SSB protein was purified by metal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized by fluorescence titration experiments for single-stranded DNA binding activity. We have applied the use of His-tagged SSB protein to increase amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving PCR efficiency.

Amino Acid Sequence↗