Enhancing PCR amplification and sequencing using DNA-binding proteins

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility

The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.     

Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase.

Is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? To identify intermolecular associations among deoxyribonucleotide biosynthetic enzymes and to ask whether these enzymes are linked to replication proteins, we analyzed radiolabeled T4 bacteriophage proteins that bind specifically to a column of immobilized T4 deoxycytidylate hydroxymethylase. More than a dozen T4 proteins and a few Escherichia coli proteins are adsorbed specifically by this column. Several of the T4 proteins were identified by two-dimensional gel electrophoresis and radioautography. These include five enzymes involved in DNA precursor biosynthesis, dCMP hydroxymethylase, thymidylate synthase, dihydrofolate reductase, dCTPase-dUTPase, and ribonucleotide reductase large and small subunits, plus several proteins of DNA metabolism and replication. Analysis of extracts of cells infected with phage amber mutants defective in specific proteins suggested a specific association involving thymidylate synthase and the gene 32 single-strand DNA-binding protein.     

A picoliter chamber array for cell-free protein synthesis

The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.     

Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity.

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.     

A lateral flow biosensor for rapid detection of DNA-binding protein c-jun

A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity.     

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.     

Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.     

Eukaryotic HMGB proteins as replacements for HU in E. coli repression loop formation

DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.     

Minimal Core Domain of HIV-1 Integrase for Biological Activity

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.     

Detecting protein-protein interactions using Renilla luciferase fusion proteins

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Detection and comparison of protein-DNA interactions using DNA-BIND plate and horseradish peroxidase-based colorimetric assay

We describe a procedure for detection and comparison of protein–DNA interactions using DNA–BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA–BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein–DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins.     

Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?

The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study. This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli. Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions. However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain. Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose. Investigation of this loss of binding suggests that LacR requires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA. In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices. Dimeric LacR variants will only bind if they have certain C-terminal extensions. These experiments sound a cautionary note for the use of filter binding as an assay of choice, particularly in applications involving screening for the DNA-binding site of putative DNA-binding proteins.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.