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PubMed1991

A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active.

We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase.

Amino Acid Sequence↗
PubMedDate not supplied

Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage.

A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.

Amino Acid Sequence↗
PubMed1992

Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81.

A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered. Cotransfection of CC81 cells with infectious BLV DNA and pAGR led to effective inhibition of BLV replication by the antisense RNA, evidenced by a drop in the number of syncytia and reverse transcriptase activity. Maximal inhibition of BLV replication (95-97%) was observed at a weight ratio of input viral and plasmid DNAs equal to 1:10.

Animals↗
PubMed2026

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗
PubMed1985

Identification and isolation of human insulin A and B chains by high-performance liquid chromatography.

A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.

Amino Acids↗
PubMed1988

Disulfide bonds and thermal stability in T4 lysozyme.

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.

Disulfides↗
PubMed1990

[Direct-action mutagens in exhausts of vehicles with diesel engines].

The genotoxic activity of exhausts from one-shaft gas-turbine GTE-5 engine (30 kW) and a standard D-54A diesel (40 kW) have been studied. Thus, the extracts of soot from GTE-5 and D-54A induced reversions in Salmonella typhimurium both with and without metabolic activation: furthermore, extracts of soot from GTE-5 demonstrated a higher mutagenic activity. The direct mutagenic effect of the exhausts depended neither on the presence of BP nor on the other polycyclic aromatic hydrocarbons (PAHs). Most probably, it was connected with the presence of nitro-PAHs. The need for studying the PAH content in vehicle engines' exhausts and for taking into account their effect in the control and standardization is established.

Benzopyrenes↗
PubMed1992

Vaccines for control of fertility and hormone dependent cancers.

Two vaccines, namely one inducing antibodies against hCG and the other against GnRH, are now in clinical trials. The hCG vaccine has entered Phase II clinical trials in three centres in India after successfully completing Phase I clinical studies in several centres in India and in four countries abroad. The vaccine was found to be devoid of side-effects; its effect was reversible. The available data on 179 cycles indicate that the vaccine prevents pregnancy at antibody titres above 50 ng/ml. A genetically engineered version of the vaccine has also been approved for trials in human lung cancer patients of the type which make hCG. hCG is observed to be a growth factor for such tumours. The GnRH vaccine is usable in both males and females as the deca-peptide is common to both sexes. Following suitable experimental and toxicology studies, the vaccine is currently in Phase I/Phase II clinical trials in patients of prostate carcinoma. Where antibody GnRH antibodies were induced, the LH, FSH and testosterone levels declined. This was accompanied by a reduction in prostate specific antigen. Clinical improvement was observed in many cases. The vaccine has also entered Phase I clinical studies in postpartum women, with the objective to extend the lactational amenorrhoea and extend inter-child interval.

Chorionic Gonadotropin↗
PubMedDate not supplied

[Implantation of a mutant of Escherichia coli requiring diaminopimelic acid in the digestive tract of gnotobiotic mice].

A DAP- auxotroph mutant of Escherichia coli DP50 requiring DAP and thymidine for growth was used as the receptor strain in genetic engineering. It failed to be implanted in axenic mice. However, when an inoculum containing more than 10(7) bacteria/ml was used, the DAP+ reverse mutant devoid of requirement for DAP became implanted. When axenic mice were previously associated with Clostridium difficile containing DAP in the cell wall, the strain DAP- became implanted even when the inoculum was too small to permit implantation in axenic mice. Conversely, C. butyricum and C. perenne, whose cell walls also contain DAP, did not allow the establishment of a DAP- mutant. In animals associated with complex human flora without enterobacteria, neither of the 2 DAP- and DAP+ mutants became implanted.

Amino Acids, Diamino↗
PubMed2026

A Novel Approach to Engineering Tomato Spotted Wilt Virus Infectious Clones by Disarming Key Nodes in Antiviral Defenses.

Tomato spotted wilt virus (TSWV) is an economically devastating pathogen that rapidly overcomes genetic resistance in major crops. Reverse genetic systems are crucial for investigating plant-virus interactions and resistance-breaking mechanisms, and developing these tools for segmented ambisense RNA viruses remains a crucial challenge. Current TSWV clones rely on extensively modified Asian isolates requiring co-delivery of multiple replication helpers and viral silencing suppressors. Streamlining these systems for regionally significant strains with minimal genetic alterations is essential. Here, we developed the first infectious clone of a U.S. TSWV isolate (PA01). Three binary plasmids contain cDNAs for the antigenomic L and S segments, as well as the genomic M segment, with enhanced GFP replacing NSs on the S segment. Co-delivery of the cucumovirus 2b alone or in combination with tombusvirus P19 or begomovirus AL2 achieved a high proportion of systemically infected Nicotiana benthamiana and Capsicum annuum plants. In N. tabacum, co-delivering the Caenorhabditis elegans cell death suppressor CED-9 or using NahG transgenic plants produced 30 to 33% systemically infected plants. Co-delivery of 2b boosted infection levels in NahG plants to 62%. These data indicate that in addition to the antiviral RNA-silencing machinery, additional host defense pathways influence TSWV rescue and systemic infection from cDNA. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tospovirus↗
PubMed2027

Molecular Cloning and Reverse Genetics.

This chapter describes a detailed molecular biology protocol for introducing specific point mutations into the chikungunya virus (CHIKV) genome using a reverse genetics strategy. The method utilizes an overlapping PCR-based approach to generate a mutated DNA fragment, which is then cloned into a pre-engineered CHIKV infectious clone plasmid. The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector. It also includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing to confirm the mutation, and plasmid purification via miniprep. The document is structured with a clear introduction, a list of all required reagents and equipment, and a step-by-step methods section.

Cloning, Molecular↗
PubMed2026

Interface Excitons in van der Waals Sandwich Heterostructures.

Exciton engineering in van der Waals heterostructures (vdWHs) is essential for next-generation optoelectronics, yet they normally require near-perfect stacking and are highly sensitive to moiré potentials. Here, we demonstrate a polarity-engineering strategy using a γ-InSe/transition metal dichalcogenide/γ-InSe sandwich heterostructure. The out-of-plane spontaneous polarization of γ-InSe intrinsically breaks interfacial inversion symmetry, giving rise to interface excitons (IFXs) that exhibit a linear Stark effect with an ultrasmall dipole moment of 0.15 e·nm. First-principles calculations and Kelvin probe force microscopy reveal asymmetric interfacial charge transfer governed by γ-InSe's polarity. Transient spectroscopy shows nonmonotonic relaxation dynamics, including a characteristic signal reversal that indicates pre-existing interfacial charge states. Our results establish that exciton dipole moments, interlayer coupling, and relaxation dynamics can be precisely tuned through material polarity and thickness. Polarity engineering thus provides a versatile and robust route to control excitonic properties in vdWHs, offering expanded design strategies for advanced excitonic and optoelectronic devices.

Stark effect↗
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↗
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↗