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Phospholipases and their industrial applications.

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions--white biotechnology is a strong alternative.

Animals↗

Diphtheria toxin-related cytokine fusion proteins: elongation factor 2 as a target for the treatment of neoplastic disease.

We have used protein engineering and recombinant DNA methodologies in order to construct a fusion protein in which human interleukin-2 (IL-2) is genetically linked to the catalytic and transmembrane domains of diphtheria toxin. The fusion toxin, DAB486IL-2, is highly cytotoxic for only those cells which display the high affinity interleukin-2 receptor (IL-2R) on their surface. In phase I/II clinical studies the intravenous administration of DAB486IL-2 has been found to be safe, well tolerated and may lead to the induction of durable remissions in patients presenting with a variety of IL-2R positive lymphomas.

Antineoplastic Agents↗

Conformational behavior of chemically reactive alanine-rich repetitive protein polymers.

The synthesis of protein-based polymers with controlled conformational properties and functional group placement offers many opportunities for the design of advanced materials. In this work, protein engineering methods have been used to produce repetitive alanine-rich protein polymers with the sequence [(AAAQ)(5)(AAAE)(AAAQ)(5)](x) (x = 2 and 6); these macromolecules may mimic architectural features of certain alanine-rich helical sequences found in natural proteins. Various proteins from this family can be readily expressed and purified from Escherichia coli. Circular dichroic spectroscopy (CD) characterization demonstrates that the purified proteins are highly helical under a variety of conditions. Thermal analysis of [(AAAQ)(5)(AAAE)(AAAQ)(5)](2) via differential scanning calorimetry (DSC) and CD indicates that the protein undergoes a reversible helix-coil transition at approximately 45 degrees C and that the protein conformation can be manipulated at elevated temperatures depending on solution conditions. The demonstrated conformational properties of these artificial proteins suggest that they may be excellent candidates for elucidating structure-function relationships in biopolymers for nanotechnology and biological applications.

Alanine↗

Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fusion proteins.

Cytokines such as interleukin-2(IL-2), gamma interferon (IFN-gamma) and alpha tumor necrosis factor (TNF-alpha) are important mediators in immune responses against tumors. However, their therapeutic efficacy and clinical utilities in treatment of human malignancies are in large part limited due to the low concentrations of cytokine in tumors and the severe toxic side-effects derived from high-dose administration of cytokines. One critical issue to improve therapeutic efficacy is how to increase the local concentration of cytokine in tumors without causing severe side-effects. A series of recent reports demonstrated that the introduction of cytokine genes into tumor cells and subsequent local secretion can circumvent the limitations associated with the systemic cytokine administration. An alternative means of cytokine delivery is to target cytokines to tumor cells with tumor specific antibodies. Thereby, effective local cytokine concentrations can be achieved at the tumor sites without resorting to patient-specific therapy. With the advance in biotechnology, two structurally disparate domains of immunoglobulin and cytokine can be brought together into one fusion protein molecule by protein engineering These engineered antibody-cytokine fusion proteins combine the unique targeting ability of tumor-specific antibodies with the multifunctional activity of cytokines. In general, there are two commonly engineered fusion proteins, the F(ab')2/cytokine expressed in mammalian cells and the single-chain FV/cytokine expressed in Escherichia coli. Both the tumor-binding reactivity and the functional cytokine activity are maintained in most of fusion proteins. Therefore, these fusion proteins may be useful in targeting cytokine to tumors to stimulate immune destruction of tumors, while limiting severe toxic side-effects by the high dose of cytokine administration. Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing the tumor-associated antigen in vivo, and to inhibit both the primary and metastatic tumors in an immune competent animal model. Therefore, these recombinant fusion proteins may represent a new generation of novel immunotherapeutic reagents for the treatment of human malignant diseases.

Animals↗

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.

Animals↗

A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.

OBJECTIVES: The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity. DESIGN AND METHODS: A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes. RESULTS: Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found. CONCLUSION: The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.

Animals↗

Electrochemical sensor toolkit for simultaneous glutamate detection at edge of cleft and peri-soma.

Simultaneously monitoring glutamate (Glu) dynamic at edge of synaptic cleft and peri-soma is crucial for understanding Glu-related pathology. Here, we created an electrochemical Glu sensors toolkit with spatial resolution of ∼60 nm, combining biologically engineered Glu binding protein for specifically capturing Glu together with chemically designed ferrocene groups for signal labeling. Modulation conjugation approach between GluR and ferrocene significantly improved sensitivity up to 32-folds. More importantly, protein engineering of residue mutation and linker peptides flexibility expanded linear range from 10 μM to 6 mM, accelerated on/off times down to 35/40 ms. This toolkit realized real-time quantifying of Glu both at edge of cleft and peri-soma, we discovered that Glu was almost released through SLC7A11 channels in calyx of held synapse upon oxygen-glucose-deprivation, while Glu was mainly released through hemichannels upon β-amyloid42 stimulation. Our work provided a methodology for investigating Glu release and reuptake and offered insights for Glu related pathology.

Glutamic Acid↗

Developments and trends in enzyme catalysis in nonconventional media.

The conventional notion that enzymes are only active in aqueous media has long been discarded, thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure organic solvents and supercritical fluids. Enzymatic reactions in nonaqueous solvents offer new possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids and flavor esters). The use of enzymes in both macro- and microaqueous systems has been investigated especially intensively in the last two decades. Although enzymes exhibit considerable activity in nonaqueous media, the activity is low compared to that in water. This observation has led to numerous studies to modify enzymes for specific purposes by various means including protein engineering. This review covers the historical developments, major technological advances and recent trends of enzyme catalysis in nonconventional media. A brief description of different classes of enzymes and their use in industry is provided with representative examples. Recent trends including use of novel solvent systems, role of water activity, stability issues, medium and biocatalyst engineering aspects have been discussed with examples. Special attention is given to protein engineering and directed evolution.

Journal Article↗

Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.

BACKGROUND: Variants of the green fluorescent protein (GFP) with different colors would be very useful for simultaneous comparisons of multiple protein fates, developmental lineages and gene expression levels. The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent. The longest wavelengths previously reported for the excitation and emission peaks of GFP mutants are 488 and 511 nm, respectively. RESULTS: Additional substitutions, mainly in residues 145-163, have improved the brightness of the blue-shifted GFP mutants with histidine and tryptophan in place of tyrosine 66. Separate mutations have pushed the excitation and emission peaks of the most red-shifted mutant to 504 and 514 nm, respectively. At least three different colors of GFP mutants can now be cleanly distinguished from each other under the microscope, using appropriate filter sets. A fusion protein consisting of linked blue- and green-fluorescent proteins exhibits fluorescence resonance energy transfer, which is disrupted by proteolytic cleavage of the linker between the two domains. CONCLUSIONS: Our results demonstrate that the production of more and better GFP variants is possible and worthwhile. The production of such variants facilitates multicolor imaging of differential gene expression, protein localization or cell fate. Fusions between mutants of different colors may be useful substrates for the continuous in situ assay of proteases. Demonstration of energy transfer between GFP variants is an important step towards a general method for monitoring the mutual association of fusion proteins.

Amino Acid Sequence↗

Multiple sequence alignment in HTML: colored, possibly hyperlinked, compact representations.

Protein sequence alignments are widely used in protein structure prediction, protein engineering, modeling of proteins, etc. This type of representation is useful at different stages of scientific activity: looking at previous results, working on a research project, and presenting the results. There is a need to make it available through a network (intranet or WWW), in a way that allows biologists, chemists, and noncomputer specialists to look at the data and carry on research--possibly in a collaborative research. Previous methods (text-based, Java-based) are reported and their advantages are discussed. We have developed two novel approaches to represent the alignments as colored, hyper-linked HTML pages. The first method creates an HTML page that uses efficiently the image cache mechanism of a WWW browser, thereby allowing the user to browse different alignments without waiting for the images to be loaded through the network, but only for the first viewed alignment. The generated pages can be browsed with any HTML2.0-compliant browser. The second method that we propose uses W3C-CSS1-style sheets to render alignments. This new method generates pages that require recent browsers to be viewed. We implemented these methods in the Viseur program and made a WWW service available that allows a user to convert an MSF alignment file in HTML for WWW publishing. The latter service is available at http:@www.lctn.u-nancy.fr/viseur/services.htm l.

Amino Acid Sequence↗

Designing enzymes for use in organic solvents.

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.

Binding Sites↗

Energetic implications for protein phosphorylation. Conformational stability of HPr variants that mimic phosphorylated forms.

The HPr protein from Bacillus subtilis is a key protein in the phosphoenolpyruvate-sugar transport system. HPr has two biological phosphorylation sites. The active site histidine is transiently phosphorylated in the phosphotransferase reaction while phosphorylation of serine 46 diminishes the activity of HPr. Here, we use protein engineering and equilibrium protein folding experiments to determine if the two phosphorylation events are energetically coupled. Our approach is to use structural mimics of the two phosphorylated forms of HPr, where histidine 15 is replaced by a negatively charged glutamate and serine 46 is changed to an aspartate, both alone and in combination. The thermodynamic analysis of the differences in conformational stability between the single and double mutants shows that the two phosphorylation sites are not energetically coupled in the HPr protein. We also show that single mutants of the active site histidine residue can have dramatic effects on the conformational stability of HPr. Combined with structural information, the method employed here will be of general use in unraveling the biological effects of phosphorylation on protein activity.

Bacillus subtilis↗

The origami of thioredoxin-like folds.

Origami is the Japanese art of folding a piece of paper into complex shapes and forms. Much like origami of paper, Nature has used conserved protein folds to engineer proteins for a particular task. An example of a protein family, which has been used by Nature numerous times, is the thioredoxin superfamily. Proteins in the thioredoxin superfamily are all structured with a beta-sheet core surrounded with alpha-helices, and most contain a canonical CXXC motif. The remarkable feature of these proteins is that the link between them is the fold; however, their reactivity is different for each member due to small variations in this general fold as well as their active site. This review attempts to unravel the minute differences within this protein family, and it also demonstrates the ingenuity of Nature to use a conserved fold to generate a diverse collection of proteins to perform a number of different biochemical tasks.

Amino Acid Motifs↗

Engineered zinc finger proteins for controlling stem cell fate.

Stem cells are functionally defined as progenitor cells that can self-renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFP-TFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP-TFs that target the mouse Oct-4 gene (which is a major regulator of ES cell pluripotency and self-renewal). These designed transcription factors were able to regulate the transcription of Oct-4, affecting the expression of downstream genes and thus regulating ES cell differentiation.

Animals↗

[Protein- and peptide-derived radiopharmaceuticals].

Recent development of radiochemistry has provided various reagent that allow stable attachment of radiolabels to proteins in vivo. The advent of genetic and protein engineering have generated proteins and peptides with reduced or no immunogenicity or more favorable distribution characteristics. However, localization of radioactivity in nontarget tissues still constitutes a problem to be resolved for accurate diagnosis and effective therapy using the radiopharmaceuticals. Recent studies have indicated that radiometabolites after lysosomal proteolysis of proteins play a critical role in the radioactivity levels in nontarget tissues. Furthermore, radiochemical design of polypeptides that liberates radiometabolites of rapid urinary excretion after lysosomal proteolysis in nontarget tissues has facilitated rapid radioactivity elimination from nontarget tissues to urine. From these findings, we believe a new era of protein- and peptide-derived radiopharmaceuticals much more suitable for diagnostic and therapeutic applications will be opened soon.

Animals↗

Protein flexibility and aggregation state of human epidermal growth factor. A time-resolved fluorescence study of the native protein and engineered single-tryptophan mutants.

A time-resolved fluorescence spectroscopic study of the recombinant human epidermal growth factor (hEGF), a bis(tryptophan)-containing protein (Trp49-Trp50), and of the two single-tryptophan-containing engineered mutants with Trp49 or Trp50 replaced by Phe ([W49F]hEGF, [W50F]hEGF), was undertaken in order to gain insight into the conformational dynamics of the C-terminal region. Quite different position-dependent microenvironments for the two Trp residues are shown by comparing the fluorescence intensity decay of both mutants. Trp50 in the single-tryptophan mutant [W49F]EGF probably undergoes a dominant interaction with the solvent. A more heterogeneous environment of Trp49 in the [W50F]hEGF mutant is found. Moreover, the fluorescence decay of the native hEGF is not simply the additive result of the decays of both mutants: the Trp2 sequence confers a conformation of the C-terminal sequence which is more in contact with the rest of the protein molecule. By contrast, the fluorescence anisotropy decay of the native protein is quite similar to that of the single-tryptophan mutants. A high degree of rotational freedom in the C-terminal region of the protein is demonstrated. The resonance energy transfer, which could contribute to the anisotropy decay, appears therefore not to be highly efficient with respect to the depolarization motions. In addition to these local conformational and dynamic aspects of the hEGF C-terminal sequence, the fluorescence anisotropy decay data demonstrate the existence of a dimerization process of the native protein which is dependent on pH and protein concentration. This phenomenon influences the excited-state lifetime profiles and, therefore, the local conformational equilibrium of the C-terminal region.

Epidermal Growth Factor↗

Applying genetic engineering to the structural analysis of proteins.

Genetic engineering offers many techniques that can be applied to the structural analysis of proteins. These techniques aid in the characterization of the protein but also can be applied to the generation of completely new diagnostic and therapeutic agents.

Amino Acid Sequence↗

Expression and radiolabeling of recombinant proteins containing a phosphorylation motif.

Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [gamma32P]ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Adenosine Triphosphate↗