Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.     

Microbiological titration of proteins and of single amino acid content in biological materials without purification and hydrolysis

A method is described for the microbiological determination of the protein content of biological materials. This method can also be adopted to titrate the concentration of a single amino acid in the protein and has the following advantages: (1) titration can be done without purification and hydrolysis of proteins; (2) the titration graph is a straight line between 25 and 800 microgram/ml; (3) protein values agree with those obtained using the Kjeldhal method; and (4) each mutant requiring one amino acid may be used to titrate the concentration of a single amino acid of the protein. The leucine content of various kinds of flour was measured with this system.     

Introduction of protein kinase recognition sites into proteins: a review of their preparation, advantages, and applications.

Labeled proteins are used in a variety of applications. This review focuses on methods that utilize genetic engineering to introduce protein kinase recognition sites into proteins. Many protein kinase recognition sites can be introduced into proteins and serve as useful tags for a variety of purposes. The introduction of protein kinase recognition sites into proteins can be achieved without modifying the essential structure or function of the proteins. Because proteins modified by these procedures retain their activity after phosphorylation, they can be used in many applications. The phosphorylatable proteins can be labeled easily to high specific activity with radioisotopes ((32)P, (33)P, or (35)S), or the nonradioactive (31)P can be used. The use of these radioisotopes provides a convenient and safe method for radiolabeling proteins. Moreover, the use of the nonradioactive (31)P with protein tyrosine kinase recognition sites permits the tagging of proteins and their detection with the many anti-phosphotyrosine antibodies available. Overall, the procedure represents a convenient, safe, and efficient method to label proteins for a variety of applications.     

Crystals of TELSAM–target protein fusions that exhibit minimal crystal contacts and lack direct inter-TELSAM contacts

While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM–target protein fusions, as follows. (i) A TELSAM–target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM–target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM–target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM–target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Evaluation of direct effects of protein ubiquitylation using computational analysis

Ubiquitylation is an important regulatory mechanism in the eukaryotic cell. A large volume of experimental data on protein ubiquitylation has been acquired in recent years. Particular ubiquitylated lysine residues were also identified. This allows us to analyze co-localization of ubiquitylation sites and functionally important protein domains, following the idea that ubiquitylation can directly affect protein functional activity. Computational analysis suggests that ubiquitylation can affect the functional activity of some proteins through direct steric effects. (1) Ubiquitylation can block protein functional domains/active site or cause accessibility limitations. It also (2) causes steric disturbances for homo-oligomerization and (3) influences heterologous protein interactions, impeding the binding of target protein with its partners. (4) Interaction with partner proteins can be disturbed by restricted conformational flexibility. Any of these effects will result in a decrease of target protein activity. Thus, we suggest a new “loss-of-function” mechanism of protein regulation by ubiquitylation.     

Split Inteins介导的多肽链两端标记

多肽链多位点特异性标记有助于了解蛋白质的结构与功能,特别是在蛋白质的动态构象研究方面. 但是,现有的多肽链多位点特异性标记方法各有局限性,并且种类有限,所以有必要开发新的多肽链多位点特异性标记方法以满足研究需求. 本文以Diub (ubiquitin dimer) 蛋白为研究对象,借助S1和S11型2种不同的断裂蛋白质内含子 (split inteins) 的蛋白质反式剪接,将含有不同荧光基团的两种小肽成功剪接至靶蛋白的两端,最终达到对靶蛋白的末端标记目的.     

How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study

The anisotropic component of the magnetic susceptibility tensor (Δχ tensor) associated with various paramagnetic metal ions can induce pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) in proteins, yielding valuable restraints in structural studies. In particular, PCSs have successfully been used to study ligands that bind to proteins tagged with a paramagnetic metal ion, which is of great interest in fragment-based drug design. To create easy-to-interpret PCSs, the metal ion must be attached to the protein in a rigid manner. Most of the existing methods for site-specific attachment of a metal tag, however, result in tethers with residual flexibility. Here we present model calculations to quantify the extent, to which mobility of the metal-binding tag can compromise the quality of the Δχ tensor that can be determined from the PCSs observed in the protein. Assuming that the protein can be approximated by a sphere and the tag is attached by a single tether, the results show that a single effective ?χ tensor can describe the PCSs and RDCs of the protein spins very well even in the presence of substantial tag mobility, implying that PCSs of ligands in binding pockets of the protein can be predicted with similar accuracy. In contrast, the quality of the PCS prediction for nuclear spins positioned above the surface of the protein is significantly poorer, with implications for studies of protein–protein complexes. The simulations probed the sensitivity of the effective Δχ tensor to different parameters, including length of the tether between protein and metal ion, protein size, type and amplitude of tag motion, tensor orientation relative to the protein and direction of tag motion. Tether length and amplitude of motion were identified as two key parameters. It is shown that the amplitude of tag motions cannot be quantified by simple comparisons of the effective Δχ tensor with the alignment tensor determined from RDCs.     

Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen

Erythrocytes bearing the Rh(D) antigen have an Mr 30,000 integral membrane protein which can be surface-labeled with 125I and can be quantitatively immunoprecipitated from Triton X-100-solubilized spectrin-depleted membrane vesicles. The 125I-labeled Rh(D)-associated protein was purified to radiochemical homogeneity from membrane skeletons solubilized in sodium dodecyl sulfate and urea by hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The Rh(D)-associated protein was purified nearly 200-fold from 2 units of erythrocytes from DD individuals by employing similar methods on a large scale using the purified 125I-labeled Rh(D)-associated protein as a tracer. The product appeared to be greater than 95% pure and migrated as a diffuse band of Mr approximately 30,000-32,000 on silver-stained sodium dodecyl sulfate electrophoresis gels poured from 12% acrylamide. It is estimated that the Rh(D)-associated protein makes up approximately 0.5% of the original membrane protein. When concentrated, partially purified Rh(D)-associated protein forms dimers and larger oligomers which are stable in sodium dodecyl sulfate and urea. The Rh(D)-associated protein was protected from degradation when intact erythrocytes or inside out membrane vesicles were enzymatically digested. These studies indicate that the Mr 30,000 protein associated with the Rh(D) antigen is linked to the membrane skeleton, resides within the lipid bilayer with minimal extra- or intracellular protrusions, exists normally as an oligomer, and can be purified in denatured form.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli.

The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.     

Economic comparison of multiple techniques for recovering leaf protein in biomass processing

Leaf protein concentrates (LPC) can be used as a valuable co-product to cellulosic biofuel production and can also mitigate the food versus fuel controversy. Two major approaches have been considered for LPC production: a well-characterized mechanical pressing method and a less studied method involving aqueous extraction with recovery using ultrafiltration. Experimental results with switchgrass extracts show low protein recovery after filtration, particularly if protein is recovered after cellulose hydrolysis. Economic modeling suggests that aqueous extraction costs less than mechanical pressing, but due to lower protein yields and lower quality, overall profit is higher for mechanical pressing versus aqueous extraction ($26/Mg feedstock vs. $14/Mg). If modest improvements can be made in extraction yields, filtration recovery, and protein quality, then the profitability of the aqueous extraction approach can be increased to $37/Mg feedstock. This study suggests that aqueous extraction is a viable alternative for LPC co-production in a biorefinery if key improvements can be made in the process.     

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.     

Evaluation of direct effects of protein ubiquitylation using computational analysis

Ubiquitylation is an important regulatory mechanism in the eukaryotic cell. A large volume of experimental data on protein ubiquitylation has been acquired in recent years. Particular ubiquitylated lysine residues were also identified. This allows us to analyze co-localization of ubiquitylation sites and functionally important protein domains, following the idea that ubiquitylation can directly affect protein functional activity. Computational analysis suggests that ubiquitylation can affect the functional activity of some proteins through direct steric effects. (1) Ubiquitylation can block protein functional domains/active site or cause accessibility limitations. It also (2) causes steric disturbances for homo-oligomerization and (3) influences heterologous protein interactions, impeding the binding of target protein with its partners. (4) Interaction with partner proteins can be disturbed by restricted conformational flexibility. Any of these effects will result in a decrease of target protein activity. Thus, we suggest a new “loss-of-function” mechanism of protein regulation by ubiquitylation.

     

UPA, a universal protein array system for quantitative detection of protein–protein, protein–DNA, protein–RNA and protein–ligand interactions

Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.     

A new set of small,extrachromosomal expression vectors for Dictyostelium discoideum

A new set of extrachromosomal Dictyostelium expression vectors is presented that can be modified according to the experimental needs with minimal cloning efforts. To achieve this, the vector consists of four functional regions that are separated by unique restriction sites, (1) an Escherichia coli replication region, and regions for (2) replication, (3) selection and (4) protein expression in Dictyostelium. Each region was trimmed down to its smallest possible size. A basic expression vector can be constructed from these modules with a size of only 6.8 kb. By exchanging modules, a large number of vectors with different properties can be constructed. The resulting set of vectors allows most basic expression needs, such as immuno blotting, protein purification, visualization of protein localization and identification of protein–protein interactions. In addition, two genes can be simultaneously expressed on one vector, which yields far more synchronous levels of expression than when expressing two genes on separate plasmids.     

Nτ-methylhistidine – An index of the true rate of myofibrillar degradation? An appraisal

The use of N^τ-methylhistidine excretion as an index of myofibrillar protein breakdown is reviewed. It is suggested that several criteria should be considered before the technique can be considered valid and these include (i) there should be no reutilization of His (τMe) during protein synthesis, (ii) there should be little change in the His (τMe) content of the muscle during development, (iii) the metabolism, if any, of the His (τMe) should be minimal, (iv) the diet should contain no His (τMe), (v) there should be no other significant source of His (τMe) in the animal other than myofibrillar protein. Following consideration of these factors and of the data obtained using the technique, it is concluded that, with caution, it can be considered a valuable tool in the study of myofibrillar protein breakdown.