Research Tools : Synthesis & purification


Enhanced Endotoxin Detection: New Advantages in Liquid Crystal Assays for Gram-Negative Pathogens

UW–Madison researchers have now demonstrated enhanced endotoxin detection in the presence of masking agents in their previous liquid crystal system.

Unlike the LAL assay, the LC-based method does not suffer from LER or any loss of sensitivity due to the presence of cations (e.g., Ca2+ or Mg2+), buffers (e.g., citrate), surfactants (e.g., SDS), chelating agents (e.g., EDTA), proteins or nucleic acids (e.g., DNA or RNA). Thus, the LC-based method provides faster and cheaper detection of endotoxin when compared to existing methods, such as the LAL assay.

Oxygen-Responsive Bacterial Gene ‘Switch’ for Biofuel Production and More

UW–Madison researchers have taken a known promoter sequence and developed several novel variants capable of modulating downstream expression levels over a 50-fold range.

The new promoters are exquisitely sensitive to changes in O2 concentration as well as other growth conditions applicable to various industrial fermentations. These include changes in the redox state of the cell and also membrane perturbations/stress caused by production or export of hydrophobic compounds, biofuel precursors or recombinant proteins.

Some of the promoters act as tightly regulated on/off gene switches while others offer a more graded or linear response.

New Protein Production Strategy for Plants

UW–Madison researchers have identified a new plant viral IRES that can facilitate the efficient expression of multiple proteins from a single mRNA. The researchers discovered the new IRES in the Triticum mosaic virus (TriMV), a wheat virus that expresses 10 proteins from a single mRNA strand.

Phosphine Ligands Made Cheaper, Better

UW–Madison researchers have developed methods for synthesizing novel classes of chiral phosphine ligands via enantioselective copper-catalyzed halogenation. The process is rapid and flexible, and also can be used to streamline the preparation of known phosphines.

The researchers previously described their ‘recycling’ method for use with aromatic compounds. Now, they have rendered the process enantioselective using an asymmetric bidentate phosphine ligand to produce scaffolds with high enantiomeric purity.

In essence, the use of the phosphine ligand helps form a chiral center in a complex product that is otherwise costly or impossible to create.

Protecting Groups for Boronic Acids

UW–Madison researchers have developed two novel protecting groups for boronic acids that make them easier to synthesize, manipulate and use. Protecting groups prevent undesired reactions.

Compound 1 is a divalent ligand to protect hemiboronic acids (e.g., benzoxaborole). Compound 2 is a trivalent ligand that complements another popular trivalent protecting group called MIDA (N-methyliminodiacetic acid). The protected boronic acids are liberated by aqueous acid.

Boronic acids treated with either of the protecting groups become highly luminescent when exposed to long-wave UV light, facilitating their use.

Concentrating Dairy Proteins

UW–Madison researchers have developed negatively charged ultrafiltration membranes for improved concentration of milk casein, whey and serum dairy proteins.

The membranes are fabricated from commercial membranes having pore sizes traditionally thought to be too large. The surface of the membrane is modified to permanently attach a negative charge that repels proteins. Taken together, the increased pore size allows higher permeability of liquid through the membrane while the negative charge helps prevent protein loss. The negative surface also is antifouling, making cleaning easier and more sustainable.

Translation-Coupling Cassette for Quickly and Reliably Monitoring Protein Translation in Host Cells

UW–Madison researchers have developed a method of using translation coupling to quickly and reliably determine whether a given host is capable of expressing the gene product of any given gene. This method could be used to monitor protein translation efficiency in bacterial cells.

The method involves a cassette that couples the full translation of a desired target gene to that of a detectable response gene. If the target gene is fully translated, so is the response gene. If the target gene is not translated, the response gene product is not detectable.

For example, one embodiment utilizes a DNA plasmid with a cloning site upstream of a DNA hairpin that masks the ribosome binding site required for translation of an antibiotic resistance protein. Scientists clone the protein of interest into the plasmid without a stop codon in frame and with a tag for purification purposes. Expression of the desired protein leads to unwinding of the DNA hairpin, unmasking the ribosome binding site and promoting translation of the antibiotic resistance protein. If the protein of interest is expressed in E. coli, the bacteria will survive in the presence of the antibiotic. If the protein is not expressed, the bacteria will die when exposed to the antibiotic.

Traceless Staudinger Ligation for the Synthesis of Peptides and Proteins in Water

UW–Madison researchers have discovered that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent such as bis(p-dimethylaminoethylphenyl)phosphinomethanethiol.  This discovery enables the formation of an amide bond in a physiological setting.  It integrates traceless Staudinger ligation with expressed protein ligation, thus extending the reach of modern protein chemistry.

Functionalized Substrate for Removal of Virus from Proteins, Particularly Antibodies

UW–Madison researchers have now developed additional virus-binding ligands for the adsorptive membrane or other substrate. When a virus-containing protein solution is passed through the membrane, the virus is irreversibly trapped while antibodies or other proteins pass through.

Simpler Method of Producing Functional Cytochrome B5 and Other Membrane Polypeptides

UW-Madison researchers have developed a simpler method of producing and purifying functional membrane polypeptides, such as cytochrome b5. This method utilizes a recombinant expression vector that expresses a fusion of cytochrome b5 and a solubilizing agent, such as maltose binding protein (MBP).

The vector can be used to express the fusion protein in solution. The protein optionally can be purified, and then a simple chemical or enzymatic cleavage reaction can release the MBP moiety when the functional, full length cytochrome b5 is needed.

In addition, the inventors discovered that the carboxy terminus of the cytochrome b5 peptide functions as a membrane anchor. To use this method to synthesize other functional membrane proteins that do not have a C-terminal membrane anchor, this anchor section can be fused to the protein, allowing it to spontaneously attach to a membrane when the solubilizing agent is released.

Enhancing the Production of Foreign Proteins in Yeast by Overexpressing Endogenous Genes

UW-Madison researchers have developed a method of enhancing the expression of foreign proteins in yeast. They identified five yeast genes that increase protein secretion. To improve the production of heterologous proteins, a yeast cell can be engineered to overexpress one or more of these genes. The genes work best to enhance the expression of proteins expressed at low levels due to low stability or folding efficiency.

Adsorptive Membrane for Removal of Virus from Proteins, Particularly Antibodies

A UW–Madison researcher has developed an adsorptive membrane that irreversibly binds viruses while allowing a protein of interest to pass through. Because most viruses are negatively charged at neutral pH and monoclonal antibodies are positively charged, the membrane contains a positively charged ligand that attracts virus while repelling antibody. However, because ion attraction alone is not sufficient for separation at high salt concentrations that disrupt ionic interactions, salt-tolerant, multimodal, anion-exchange ligands are used. When a virus-containing protein solution is passed through the membrane, the virus is irreversibly trapped while the antibody passes through.

Membrane Cascade-Based Separation

UW-Madison researchers have developed a counter flow cascade system that features a novel separation technique designed to reduce the cost of manufacturing high-value biological materials. The technique promises to substantially reduce capital and operating costs and is an improvement over current separation processes, including process scale chromatography and simulated moving beds. 

This system consists of a linear combination of modules forming what is known as a counterflow cascade.  Each module consists of a semipermeable membrane through which the solution to be separated is passed.  A diafiltration membrane that is selectively permeable to a first solute divides the solution into a permeate flow containing the first solute, and a retentate flow containing a second solute. After passing through the diafilter, the permeate flow passes through an ultrafiltration membrane, which allows only the solvent to pass through. The ultrafilter removes excess solvent from the permeate flow and recycles it back into the system.

Each module after the first accepts a mixed flow stream formed by combining the retentate flow and permeate flow from different stages. The flow rates are adjusted so that the first and second solutes are present in the same molar ratio in the mixed flow stream.

Singlet Oxygen-Resistant Technologies

UW-Madison researchers have developed a method of altering the response of cells to 1O2 by modulating the interaction between an anti-sigma factor, ChrR, and σE, or by altering the expression of a gene product required for viability in the presence of 1O2. The growth of phototrophic bacteria exposed to 1O2 may be inhibited by administering an anti-sigma agent, such as ChrR, to reduce the availability of σE On the other hand, a bacterium or other organism may be protected from damage from 1O2 by modifying the genes in the σE regulon or by modifying ChrR to alter binding between it and σE.

Production of Packaged DNA Sequence

UW-Madison researchers have developed a method for packaging the full papillomaviral genome into HPV virions in transfected mammalian cells, making easy and efficient generation of infectious HPV possible for the first time. A DNA sequence containing a full-length or nearly full-length papillomaviral genome is selected and preferably cloned and amplified in an appropriate plasmid. The genome sequence is then isolated from the plasmid and recircularized before it is co-transfected with an HPV capsid sequence into a suitable mammalian cell. Finally, the resulting infectious viral particles are purified. Either the papillomaviral genome or the HPV capsid sequence may be modified before packaging.

Membrane Cascade-Based Separation

A UW-Madison researcher has developed a counter flow cascade separation system for separating two solutes in a solvent. The system consists of a series of interconnected stages through which a solution flows. At each stage, a diafiltration membrane that is selectively permeable to the first solute divides the solution into a permeate flow containing the first solute, and a retentate flow containing the second solute.

After passing through the diafilter, the permeate flow passes through an ultrafiltration membrane, which allows only the solvent to pass through. The ultrafilter removes excess solvent from the permeate flow and recycles it back into the system.

Each stage after the first accepts a mixed flow stream formed by combining the retentate flow and permeate flow from different stages. The flow rates are adjusted so that the first and second solutes are present in the same molar ratio in the mixed flow stream.

Methods for Ligating Molecules to Surfaces

UW-Madison researchers have now discovered that Staudinger ligation provides a rapid and efficient means to covalently attach proteins or other molecules to a specific site on a surface. The covalent ligation is based on the reaction of an azide and a phosphinothioester to form an amide bond. Preferably, a phosphinothioester is covalently bound to the surface through a linker group, and an azide functional group is attached to the molecule to be ligated. Alternatively, the azide group can be linked to the surface and the phosphinothioester can be attached to the molecule to be ligated. Next, the molecule to be immobilized is added to the surface so the phosphinothioester and the azide group can react to form an amide bond, covalently linking the molecule to the surface.

Heterogeneous Protein Foldamers Containing Alpha, Beta and Gamma Amino Acids

UW-Madison researchers have developed polypeptide foldamers containing alpha-amino acids along with cyclically constrained beta-amino acids and gamma-amino acids. These unnatural compounds contain rotationally constrained amino acid residues that are not amenable to enzymatic degradation, making them useful to probe protein-protein and other large molecule interactions. Because the backbone is heterogeneous, a portion of the residues, such as the alpha-amino acids, can provide functional diversity, while the cyclically constrained residues confer conformational specificity and stability.

Chemical Synthesis of Reagents for Peptide Coupling

UW-Madison researchers have developed improved methods for synthesizing phosphinothiol reagents. These synthesis techniques take advantage of phosphorus’s strong interaction with boron, and are based on an easily prepared alkylating agent and a commercially available borane-organophosphine complex. In the methods, the alkylating agent is reacted with the borane-organophosphine complex. Disrupting the resulting phosphine-borane complex and removing the protecting group generates the phosphinothiol reagents.

Method of Error Reduction in Nucleic Acid Populations

UW-Madison researchers have developed a method for proofreading oligonucleotides synthesized and eluted from the glass surface of an array and eliminating those oligonucleotides containing errors. During synthesis of oligonucleotides on a microarray surface, a complementary copy of the oligonucleotide is made. When the single-stranded molecules are eluted from the surface, the complementary strands hybridize to form short double-stranded molecules. If errors are present in any of the shorter strands, they will not yield perfectly matched duplexes. These mismatched duplexes can be detected and eliminated by various means.

Staudinger Ligation Method for Rapid and Reliable Chemical Synthesis of Proteins

UW-Madison researchers have developed an improved method of synthesizing proteins that does not require a cysteine residue at the ligation junction.  This method expands the utility of total protein synthesis by removing the limitation inherent in native chemical ligation and expressed protein ligation.  It is inspired by the Staudinger reaction, in which a phosphine molecule is used to reduce an azide to an amine. 

In this method, a phosphinothioester and an azide are united to form an amide bond.  A phosphinothiol reagent is used to efficiently generate a phosphinothioester from an amino acid, peptide or protein fragment.  An azido group then can be formed at the N-terminus or a basic side group of another amino acid, peptide or protein.  Then the phosphinothioester is reacted with the azido group to ligate the amino acids, peptides or proteins.

This reaction allows the formation of an amide bond among a wide variety of chemical species.  It can be used to repeatedly ligate natural and/or nonnatural amino acids to synthesize proteins or peptides.  Large peptides or proteins can be formed by ligating two or more small peptides or proteins.

Method and Device for Separating Particles By Size

UW-Madison researchers have developed a method and device for separating mixed particles of different sizes by using ultrasound. The invention achieves bead chromatography (separation by size) by using acoustic radiation force in an ultrasonic actuator. First, a mixture of particles is focused at the nodes of a standing pressure wave in a vessel such as a glass capillary. Then, an inertial, nonlinear force generated by transverse vibrations in the vessel separates the particles by size.

Flow Cell Synthesis of DNA Probes

UW-Madison researchers have developed an improved flow cell for use in the synthesis of arrays of DNA probe sequences and polypeptides. The flow cell precisely aligns a substrate with respect to an image former, while distributing a reagent-containing fluid through the active volume and over the active exposed surface of the flow cell.

The flow cell includes a base with a central window opening and a registration surface against which the substrate may be mounted. A press block is engaged against a gasket mounted on the active surface of the substrate to fully enclose an active volume between the press block, the peripheral walls of the gasket’s central opening and the active surface of the substrate. Channels in the press block extend to the extension openings in the gasket, allowing the flow of reagents into and out of the active volume.

Methods and Reagents for Capping Ruthenium or Osmium Carbene-Catalyzed ROMP Products

UW-Madison researchers have developed a method for the terminal attachment of functional groups to materials generated by ROMP. It involves preparing a telechelic polymer using a ruthenium or osmium carbene catalyst and a capping agent. More specifically, a monomer is polymerized in the presence of a ruthenium or osmium carbene catalyst to form a polymer template. Then the polymer template is combined with a functionalized capping agent under conditions for forming a terminally functionalized polymer.

DNA Molecules Encoding Bacterial Lysine 2,3-Aminomutase

UW-Madison researchers have developed methods to prepare L-beta-lysine using DNA molecules that encode lysine 2,3-aminomutase. Lysine 2,3-aminomutase catalyzes the reversible isomerization of L-lysine into L-beta-lysine. The researchers isolated and sequenced the gene for lysine 2,3-aminomutase from a strain of Clostridium. To prepare L-beta-lysine, host cells expressing recombinant lysine 2,3-aminomutase are cultured in the presence of L-lysine. Then the L-beta-lysine is purified from the cultured host cells. Alternatively, lysine 2,3-aminomutase is isolated from the host cells and reacted with L-lysine to produce L-beta-lysine.

Amphiphilic Agents for Membrane Protein Solubilization

UW-Madison researchers have now developed novel amphiphilic detergent molecules for solubilizing and crystallizing membrane proteins into ordered structures. The key feature of these molecules is their rigid hydrophobic domain. The domain renders the detergent molecules rigid enough to minimize intramolecular disorder during crystallization, yet sufficiently flexible to promote the protein-protein interactions that create the lattice structure in a membrane protein-detergent crystal.

Expression and Purification of Recombinant Rabbit and Human Phosphoglucomutase

UW-Madison researchers have now developed a method for expressing and purifying PGM using an E. coli system. Their technique produces high yields of recombinant PGM in a soluble, active form. A variety of materials are available for licensing, including whole cell pastes of recombinant E. coli, cell-free extracts and purified recombinant proteins.

An Epitope Tag for Gentle Immunoaffinity Chromatography

UW-Madison researchers have now developed a protein immunopurification method that allows elution of proteins from antibodies under mild conditions. To develop the technique, the researchers first selected monoclonal antibodies that released antigen under mild, non-denaturing conditions. They then identified epitope tags for the monoclonal antibodies through recombinant DNA methods.

Softag1, an epitope of a well-characterized monoclonal antibody, was identified through this method. When Softag1 was fused to proteins and expressed in E. coli, purification of the tagged proteins required only a single-step immunoaffinity chromatography procedure and elution using mild conditions.

Expression System and Fermentation Processes For Overexpression Of Holo-Acyl Carrier Protein

UW-Madison researchers have created eight bacterial expression plasmids from either E. coli or spinach, which express both apo-ACP and E. coli holo-ACP synthase, the enzyme responsible for the post-translational modification of ACP. Therefore, the plasmids allow completion of the required modification. The researchers' expression system enables the production of ACPs in high yields by using chemically defined minimal medium and specialized fermentation procedures.

Tagged RNA Polymerases and Method for Producing Them

UW-Madison researchers have developed DNA constructs to create strains of E. coli (RL916 and RL1200) that produce several different types of tagged RNA polymerases. This technique yields E. coli strains that produce RNA polymerase with tags on the C-terminus of the beta prime subunit, a position that can be derivatized without adverse effects on enzyme activity. Biochemical tags that have been added include S. aureus protein A, E. coli biotin carrier protein, green fluorescent protein and beta galactosidase.