Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.     

Controlling the orientation of immobilized proteins on an affinity membrane through chelation of a histidine tag to a chitosan-Ni surface

The ability of histidine-tagged proteins to chelate to Ni⁺⁺ ions that are coordinated to functionalized polymeric matrices is the basis of affinity column separations. In this study, affinity membranes based on a Ni⁺⁺-chelating chitosan surface were fabricated to immobilize C-terminus hexahistidine-tagged green fluorescent protein (his-GFP). The binding of GFP antibody (antiGFP) to the immobilized his-GFP was measured and compared to a membrane with a chitosan surface to which his-GFP was immobilized through amine-glutaraldehyde chemistry as a control. Both membranes had comparable amounts of his-GFP immobilized on the surface. However, the amount of antiGFP bound to the Ni⁺⁺-chelated his-GFP at saturation was higher than that bound to the glutaraldehyde-immobilized his-GFP by a factor of five. Furthermore, fitting the data to a single-site Langmuir model resulted in an affinity constant for the Ni⁺⁺-chelated his-GFP towards antiGFP that was 14 times higher than the glutaraldehyde-immobilized his-GFP. The higher affinity suggests that immobilizing a protein at its C-terminus results in the proper orientation for subsequent antibody binding. At low antibody concentrations, the sensitivity of the affinity membrane is 70 times that of the control.     

Metallization of a genetically engineered polypeptide

Recently, well-ordered biological materials have been exploited to pattern inorganic nanoparticles into linear arrays that are of particular interest for nanoelectronic applications. In this work, a de novo designed E. coli-expressed polypeptide (previously shown to form highly rectilinear, β-sheet-containing structures) operates as a template for divalent metal cations. EDX and TEM analysis verify the attachment of platinum ions to the histidine-rich fibril surface, which was designed specifically to facilitate attachment of chemical moieties. Following chemical reduction, TEM further confirms the formation of localized zero-valent metal aggregates with sub-nanometer interparticle spacing.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Point of attachment and sequence of immobilized peptide-acridine conjugates control affinity for nucleic acids

Screening of combinatorial libraries by spatial arraying strategies requires library members to be solid-phase immobilized. However, for nucleic acid ligands that bind via intercalation, immobilization may inhibit binding if the tethering functionality is present at the edge of the heterocyle that approaches the duplex during the binding reaction. We report here a method for immobilizing peptide-acridine conjugates (PACs) via either their C- or their N-terminus, corresponding to functionalization at either the 4- or the 9-position of acridine, respectively, and for assaying the nucleic acid binding properties of the resulting resins. We find that both the amino acid sequence of the PAC as well as its point of attachment to the solid support are important in determining affinity for duplex nucleic acids. These results have implications for the design of future on-bead and microarray-based selections and in understanding the nucleic acid binding of functionalized intercalators.     

Purification of recombinant proteins by chemical removal of the affinity tag

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.     

Separation of chitosan oligomers by immobilized metal affinity chromatography

A novel approach for chitosan oligosaccharide (COS) separation by immobilized metal affinity chromatography (IMAC) based on the differences in the interactions of chelated copper (II) ions with various COS (dimers, trimers, tetramers) is described. Polyhydroxylic chromatographic supports (agarose CL-6B and silica) were functionalized with various chelating functions such as iminodiacetate (IDA), carboxymethyl-aspartate (CM-Asp) and tris(carboxymethyl)ethylenediamine (TED). The COS retention capacities of the columns were between 2 and 6 mg/cm(3), depending on the chelating group. The COS were separated and/or enriched up to 95% for dimer and trimer and 90% for the tetramer, with yields of 60-95%.     

Protein glycoengineering enabled by the versatile synthesis of aminooxy glycans and the genetically encoded aldehyde tag

Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein-glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features N-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone.     

Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) was investigated as a method of recovery for green fluorescent protein (GFPuv). It was found that in the absence of genetic modification to enhance metal affinity, GFPuv displayed strong metal affinity to Cu(II) and Ni(II), and weak or negligible affinity to Zn(II) and Co(II). Changes in the mobile phase NaCl concentration during Ni(II)-IMAC strongly affected purity and yield of GFPuv, with fine resolution under higher NaCl concentrations. Finally, IMAC via Cu(II) and Zn(II) with intervening diafiltration was used to recover GFPuv with high yield and purity.     

Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.     

Fractionation of glycoprotein-derived oligosaccharides by affinity chromatography using immobilized lectin columns

Lectin affinity column chromatography is becoming a method of choice for the fractionation and purification of oligosaccharides, especially N-linked oligosaccharides. Using lectin affinity, it is easy to separate structural isomers and to isolate oligosaccharides based on specific features. Further, serial lectin column chromatography, when various lectin columns are used at the same time, can afford a very sensitive method for the fractionation and characterization of extremely small amounts of oligosaccharides. Thus, when used in conjunction with other separation techniques, lectin affinity chromatography can help to purify rapidly oligosaccharides and provide substantial information about their structural features.     

Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.     

Effects of pH and acetic acid on glucose and xylose metabolism by a genetically engineered ethanologenicEscherichia coli

Applied Biochemistry and Biotechnology - Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce...     

Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids

Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals.     

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.     

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.     

Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (～0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.     

Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins

Chelation of mercuric ions by an iminodiacetate-Sepharose gel was evaluated. The retentive properties of iminodiacetate-Sepharose gel column was studied towards proteins varying the composition of eluting systems from 2-mercaptoethanol to NaCl and imidazole, determining also the extent of mercury leaching. It was demonstrated that chelated mercury contained free sites for interaction with proteins such as bromelain and recombinant human granulocyte colony stimulating factor from E. coli. The extraction of the latter by chromatography of its inclusion bodies solution on Hg(II)-loaded Sepharose-iminodiacetate gel was also evaluated.