Methods for improved protein expression using pET vectors.
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Blotting, Western↗
Biomedical subjects
J Conner
Publications and source records attributed to J Conner.
Natural selection on body size in Tribolium: possible genetic constraints on adaptive evolution.
To determine whether genetic constraints on adaptive evolution were operating in a laboratory population of a flour beetle, Tribolium castaneum, we first estimated the direct selection acting on each of several body size traits. Strong selection in males for an increase in pupal weight and a decrease in the ratio of adult to pupal weight occurred. In addition, a non-significant trend for a decrease in adult width was found. No significant selection on females was detected, although there were trends toward an increase in pupal weight and a decrease in adult width. These estimates were then combined with estimates of the genetic variances and covariances of the traits to predict the multivariate response to selection, that is, the evolutionary change in the traits across one generation. These projections showed only a small predicted change in male pupal weight in spite of the strong selection on pupal weight, and a relatively large predicted increase in width in spite of the possible negative direct selection on this trait. Both of these results were due in part to the positive genetic covariance between pupal weight and width, and they therefore suggest the possibility of genetic constraints on adaptive evolution of these traits.
Animals↗
The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity.
Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.
Amino Acid Sequence↗
An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.
We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.
Adenosine Triphosphate↗
Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes.
Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.
Animals↗
The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification.
The open reading frame of the large subunit (R1) of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been positioned downstream of the phage T7 gene 10 promoter in the expression vector, pET. Transformation of this recombinant plasmid into Escherichia coli BL21 DE3 cells containing the T7 RNA polymerase, under the control of the lac UV5 promoter, allows expression of the subunit on induction of the T7 RNA polymerase by isopropyl thiodigalactoside. The expressed protein is soluble and can be purified with yields up to 0.5 mg of R1 per litre of bacterial culture. The subunit can complement R2 produced in BHK cells or E. coli to give specific activities comparable to that produced in BHK cells infected with HSV-1. Enzyme activity reconstituted from E. coli-expressed R1 and R2 is inhibited by the nonapeptide YAGAVVNDL with an IC50 comparable to that obtained with enzyme extracted from BHK cells infected with HSV-1. Results suggest that the E. coli produced enzyme is a good source of protein for further structural and functional studies.
Animals↗
Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli.
The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.
Amino Acids↗
Post-arthroscopic pulmonary edema in two healthy teenage athletes.
Outpatient arthroscopic knee surgery carries with it certain risk factors similar to those accompanying other major knee procedures. These risks may be related to the complexity of the procedure, exposure to anesthesia, or risk factors from previous medical history. Two cases of acute pulmonary edema following arthroscopic knee surgery in otherwise healthy teenage athletes are presented. Both patients developed acute respiratory distress in the recovery room after uneventful arthroscopic knee surgery. The patients in both cases recovered and were able to return to sporting activity with no sequelae. The similarities in both cases prompted a retrospective investigation of the events from the induction of general anesthesia to the admission of the patients to the intensive care unit. Several possible causes of acute post-operative pulmonary edema include fluid overload, cardiac arrhythmia, respiratory depression, systemic drug reaction and sickle cell trait or disease. Outpatient arthroscopy still remains the procedure of choice for meniscal pathology of the knee, but the surgeon and the anesthesia personnel must be aware of and prepared for pulmonary complications that may arise in the immediate post-operative period.
Acute Disease↗
Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.
The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.
Albumins↗