A novel means of asymmetric linker attachment to DNA molecule by RecA-mediated multistranded DNA formation

We have developed a novel asymmetric linker attachment technology that utilizes multistranded DNA formation mediated by the RecA protein and Exonuclease I. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary oligonucleotide in the presence of RecA and Exonuclease I. We have explored the possibility of applying this finding to the asymmetric attachment of linkers to specific DNA termini. We show that these unique properties of the terminal triple-stranded structure can be applied to directly clone specific DNA sequences.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.     

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (R _gfp/A _xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R _gfp/A _xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.     

Construction and characterization of a bi-functional EGFP/sBAFF fusion protein

A fusion between gene encoding fluoresce-enhanced green fluorescent protein variant (EGFP) and soluble domain of human B-cell-activating factor of the TNF family (sBAFF) was constructed and expressed in Escherichia coli. The EGFP/sBAFF had an apparent molecular weight of 45 kDa and was detected with anti-hsBAFF and anti-His(6) monoclonal antibodies. After being purified by immobilized metal affinity chromatography (IMAC), the fusion protein retained similar fluorescence spectra to those of EGFP. Biological activity assays showed the EGFP/sBAFF as well as sBAFF could co-stimulated human B lymphocyte proliferation in vitro. In addition, EGFP/sBAFF has shown specific binding to BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active sBAFF that may prove useful in further studies on BAFF and its receptors.     

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.     

Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase.

While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

The impact of protein flexibility on protein-protein docking

Accounting for protein flexibility in protein-protein docking algorithms is challenging, and most algorithms therefore treat proteins as rigid bodies or permit side-chain motion only. While the consequences are obvious when there are large conformational changes upon binding, the situation is less clear for the modest conformational changes that occur upon formation of most protein-protein complexes. We have therefore studied the impact of local protein flexibility on protein-protein association by means of rigid body and torsion angle dynamics simulation. The binding of barnase and barstar was chosen as a model system for this study, because the complexation of these 2 proteins is well-characterized experimentally, and the conformational changes accompanying binding are modest. On the side-chain level, we show that the orientation of particular residues at the interface (so-called hotspot residues) have a crucial influence on the way contacts are established during docking from short protein separations of approximately 5 A. However, side-chain torsion angle dynamics simulations did not result in satisfactory docking of the proteins when using the unbound protein structures. This can be explained by our observations that, on the backbone level, even small (2 A) local loop deformations affect the dynamics of contact formation upon docking. Complementary shape-based docking calculations confirm this result, which indicates that both side-chain and backbone levels of flexibility influence short-range protein-protein association and should be treated simultaneously for atomic-detail computational docking of proteins.     

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies

Abstract STb is a heat-stable enterotoxin elaborated by enterotoxigenic Escherichia coli strains associated with weaning piglets and is responsible for diarrhoea in those animals. The maltose binding protein (MBP) of E. coli was used as a carrier for STb, a poorly immunogenic molecule. Constructions were produced where the gene coding for mature STb toxin (MBP-STb) and a fragment of the gene spanning the major epitopic region of STb (AA8–AA30)(MBP-STb₂) were fused to malE gene coding for MBP. The fusion proteins accumulated in the periplasm and were detected with a polyclonal antibody raised against the purified toxin. MBP-STb induced secretion in the biological model whereas MBP-STb₂ was non-toxic. Immunization of rabbits evoked an antibody response to STb for these two fusion proteins. However, only MBP-STb elicited antibodies that effectively neutralized the toxicity of pure STb toxin as determined in the rat loop assay.     

Functional improvement of antibody fragments using a novel phage coat protein III fusion system

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.     

A machine learning approach for the prediction of protein surface loop flexibility

Proteins often undergo conformational changes when binding to each other. A major fraction of backbone conformational changes involves motion on the protein surface, particularly in loops. Accounting for the motion of protein surface loops represents a challenge for protein-protein docking algorithms. A first step in addressing this challenge is to distinguish protein surface loops that are likely to undergo backbone conformational changes upon protein-protein binding (mobile loops) from those that are not (stationary loops). In this study, we developed a machine learning strategy based on support vector machines (SVMs). Our SVM uses three features of loop residues in the unbound protein structures-Ramachandran angles, crystallographic B-factors, and relative accessible surface area-to distinguish mobile loops from stationary ones. This method yields an average prediction accuracy of 75.3% compared with a random prediction accuracy of 50%, and an average of 0.79 area under the receiver operating characteristic (ROC) curve using cross-validation. Testing the method on an independent dataset, we obtained a prediction accuracy of 70.5%. Finally, we applied the method to 11 complexes that involve members from the Ras superfamily and achieved prediction accuracy of 92.8% for the Ras superfamily proteins and 74.4% for their binding partners.