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PubMed1985

Immunological detection of cauliflower mosaic virus gene V protein produced in engineered bacteria or infected plants.

Antiserum was prepared against a synthetic peptide corresponding to the C-terminal 25 amino acids (aa) of the protein encoded by cauliflower mosaic virus (CaMV) gene V, which is thought to be a reverse transcriptase involved in viral DNA replication. This antiserum was used to detect the expression of CaMV gene V either in Escherichia coli JM103 transformed by an expression vector containing CaMV gene V or in CaMV-infected plants. In both cases, an 80-kDal protein has been detected.

Amino Acid Sequence↗

PubMed1992

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions.

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

3T3 Cells↗

PubMed1995

Roles of nitric oxide in tumor growth.

A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type parental cells. This growth retardation was reversed by the addition of N-iminoethyl-L-ornithine. In nude mice, however, the tumors from these cells grew faster than those derived from wild-type cells and were markedly more vascularized, suggesting that NO may act as part of a signaling cascade for neovascularization. Recent observations that the generation of NO in human breast and gynecological cancers correlates positively with tumor grade are consistent with this hypothesis. We suggest that NO may have a dual pro- and antitumor action, depending on the local concentration of the molecule.

Adenocarcinoma↗

PubMed1995

Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice.

We studied the expression of the angiogenic factor vascular permeability factor) (VPF, also called vascular endothelial growth factor), in human melanoma cells in vitro and in vivo. Melanoma lines that develop tumors with a low metastatic potential in nude mice were found to have low expression levels of VPF in vitro, and the VPF expression levels in melanoma lines that yield highly metastatic xenografts were high. However, in vivo the correlation between VPF mRNA levels and the frequency of metastasis was lost; in all xenografts equally high levels of VPF mRNA were found, independent of the parental cell line. Hence, in vivo VPF gene expression was upregulated in the low expressing lines. The external factor responsible for this induction may be hypoxia, given that we found that low oxygen tension caused a (reversible) increase in the VPF mRNA levels in otherwise low expressing melanoma lines in vitro. A melanoma line with an inducible VPF expression was engineered into a line with a constitutive VPF expression. In the xenografts from this line a change in the vascular architecture was seen, indicating that the pattern or the level of VPF expression is important for tumor angiogenesis in melanoma xenografts.

Alternative Splicing↗

PubMed1995

Exploring the peptide 3(10)-helix reversible alpha-helix equilibrium with double label electron spin resonance.

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3(10)-helix in 16-residue model peptides containing only L-amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length-dependent 3(10)-helix-->alpha-helix transition and the role of Arg+ in the stabilization of specific helix structures.

Amino Acid Sequence↗

PubMed1992

Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.

Amino Acid Sequence↗

PubMed1988

Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation.

Replacement of the putative active site Asp residue of cloned HIV-1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild-type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans-complementation of a genetically engineered gag--protease fusion protein was observed.

Amino Acid Sequence↗

PubMed1985

Visual effects with a miniature Leonardo's window: photographs and real scenes fused stereoscopically.

The drawing on a conventional Leonardo's window will be seen by both of an observer's eyes unless one of them is occluded. But, by substituting a photographic colour transparency (in a suitably compact viewer) for the drawing, one eye can view the transparency whilst the other eye gains an unobstructed direct view of the scene. The transparency can then be stereoscopically fused with the directly-viewed scene. As the perspective of the transparency depends not on the observer's viewpoint but only on the position of the camera at the time of photography, altering the viewpoint causes the effective eye-base to vary, thus enhancing, suppressing, or sometimes even reversing the stereoscopic depth. With very simple equipment several visual effects can easily be demonstrated. Practical applications of the technique have been found, particularly in those engineering and geological situations in which it is useful to detect changes since a photograph was taken.

Adult↗

PubMed2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

Animals↗

PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗

PubMed1989

Mutants of pertussis toxin suitable for vaccine development.

Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.

Animals↗

PubMed1995

Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC.

Three highly attenuated and efficacious poxvirus-based vectors, NYVAC, ALVAC and TROVAC, are available for targeted applications as recombinant vaccines in both human and veterinary medicine. The attenuated phenotype of the three vectors is consistent with safe use for vaccination purposes, for the vaccinee, for unvaccinated contacts, and for introduction into the environment. The precise deletion of virulence and host range genes in the NYVAC vector precludes reversion to the virulent phenotype by back mutation. Dissemination of recombinant vaccines based on the NYVAC, ALVAC and TROVAC vectors is highly diminished, because of the genetic engineering in NYVAC and the natural attenuated phenotype of ALVAC and TROVAC. Studies have demonstrated that these recombinant vectors are genetically and phenotypically stable after serial passage in vitro as well as in vivo. NYVAC, ALVAC and TROVAC vectors are the only three poxvirus-based vectors that are classified as BSL1 agents.

Animals↗

PubMed1995

Conversion of thrombin into an anticoagulant by protein engineering.

At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose-dependent, reversible anticoagulation without any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.

Amino Acid Sequence↗

PubMedDate not supplied

Site-directed mutagenesis for quantitation of base-base interactions at defined sites.

Two alkylation products implicated in initiation of carcinogenesis are O6-alkylguanine (m6G) and O4-alkylthymine (m4T). We have used site-specific insertion of these derivatives into oligonucleotides and measured the kinetic constants of various pairings, using both prokaryotic and eukaryotic polymerases for replication. Preliminary data are also reported for another carcinogen product, N2,3-ethenodeoxyguanosine ( epsilon G). The immediate neighbor bases play an important role in determining the frequency of specific changed basepairing and subsequent elongation of the annealed primer. However, both m4T and m6G prefer to form a type of G.T pairing which would lead to the transitions: G.C----A.T or T.A----C.G. The enzymes were the Klenow fragment of E. coli DNA polymerase I (Kf), engineered 3'----5' exonuclease-free Kf (exo-free Kf), polymerase alpha-primase complex from Drosophila melanogaster or calf thymus, and human immunodeficient virus-I reverse transcriptase (HIV-I RT). All enzymes led to approximately the same frequency of transitions. It is postulated that the mutation frequency at a given site is primarily a function of the structure of the sequence around the target site.

Animals↗

PubMed2026

Mechanisms linking the gut microbiota to colorectal cancer development and progression.

Colorectal cancer remains a leading cause of global cancer mortality, with a concerning rise in early-onset cases driven by complex interactions between environmental exposures, lifestyle factors, and host genetics. Mounting evidence indicates that gut microbiota dysbiosis critically modulates this oncogenic process, acting as an active participant rather than a passive bystander. This review systematically synthesizes the dichotomous roles of the intestinal microbiome in colorectal tumorigenesis through the conceptual framework of the driver-passenger model. We discuss how early initiating driver bacteria, such as Polyketide synthase-positive Escherichia coli and enterotoxigenic Bacteroides fragilis, compromise mucosal barriers, induce chronic mucosal inflammation, and inflict direct genomic instability. As the local tumor microenvironment undergoes profound metabolic remodeling, opportunistic passenger pathogens, notably Fusobacterium nucleatum, become enriched, further promoting cellular proliferation and facilitating tumor immune evasion. Conversely, protective commensals, exemplified by Clostridium butyricum and Streptococcus thermophilus, exert robust tumor-suppressive effects through multifaceted mechanisms. These beneficial microbes actively antagonize malignant progression by redirecting tumor metabolic fluxes toward oxidative stress, orchestrating deep epigenetic reprogramming, and degrading core oncoproteins to reverse chemoresistance. Transitioning from fundamental mechanisms to clinical application, we evaluate a comprehensive spectrum of microbiota-targeted interventions, encompassing non-invasive diagnostic biomarkers, fecal microbiota transplantation, engineered bacteria, phage therapy, and postbiotics. Finally, we critically address the formidable translational challenges associated with microbial heterogeneity, long-term safety, and regulatory standardization, aiming to provide a balanced perspective on integrating microbiome-based strategies into next-generation precision oncology for colorectal cancer.

Humans↗

PubMed1989

Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies.

The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986). This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium. The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions. The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form. The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride. These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper.

Calorimetry↗

PubMed1991

Novel guanosine requirement for catalysis by the hairpin ribozyme.

THERE is much interest in the development of 'designer ribozymes' to target destruction of RNAs in vitro and in vivo. Engineering of ribozymes with novel specificities requires detailed knowledge of the ribozyme-substrate interaction, and a rigorous evaluation of sequence specificity. The hairpin ribozyme catalyses an efficient and reversible site-specific cleavage reaction. We have used mutagenesis and in vitro selection strategies to show that RNA cleavage and ligation has an absolute requirement for guanosine immediately 3' to the cleavage-ligation site. This G is not required for efficient substrate binding, rather, its 2-amino group is an essential component of the active site required for catalysis.

Base Sequence↗

PubMed2026

Protein Language Model Decoys for Target Decoy Competition in Proteomics: Quality Assessment and Benchmarks.

Large-scale proteomics relies heavily on target-decoy competition for false discovery rate estimation in peptide identification, and the performance of this strategy depends strongly on the design of the decoy database. Classical generators such as reversal and shuffling remain widely used. Here, we introduce the first protein language model-based (PLM) decoy generation for peptide identification and benchmark it against classical strategies. We evaluate these approaches using three complementary quality-control layers: sequence-based separability, search-engine-agnostic spectral-space diagnostics, and end-to-end mass spectrometry benchmarks, including pipelines with rescoring. Across these analyses, PLM-based decoys are harder for sequence-only neural networks to distinguish than most classical generators, suggesting fewer obvious sequence-level artifacts. However, this signal is only weakly informative for search performance. Spectral diagnostics further show that short peptides occupy a particularly crowded target-decoy space and are therefore especially prone to local collisions across all generators. In full search pipelines, reverse decoys remain a strong baseline, and current PLM-based generators do not yet provide a clear overall advantage. We therefore view PLM-based decoys not as universal replacements for reverse decoys but as tunable tools for benchmarking, diagnostics, stress testing, and future adaptive decoy optimization, with increasing value as search models become more expressive.

Proteomics↗