Expression of mammalian cytochrome P450 enzymes using yeast-based vectors.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J T Ross
Publications and source records attributed to J T Ross.
Splenic spite and flawless flight.
Explore the source record for details and available documents.
Periodicals as Topic↗
L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography.
A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.
Asparaginase↗
Large-scale recovery and purification of L-asparaginase from Erwinia carotovora.
A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison.
Amino Acid Sequence↗
The large-scale isolation of bryostatin 1 from Bugula neritina following current good manufacturing practices.
A novel process was designed for the large-scale isolation of bryostatin 1 from the bryozoan Bugula neritina L. in order to obtain multigram quantities of highly pure material for formulation studies, preclinical toxicology, and clinical trials in cancer patients. Multigram quantities of bryostatin 1 were obtained from a collection of approximately 10,000 gallons of wet animal. A phorbol dibutyrate (PDBu) receptor binding assay and hplc with photodiode array detection were used for the design, validation, and control of the isolation process.
Animals↗
Complications and long-term results after partial laryngectomy for the treatment of idiopathic laryngeal paralysis in 45 dogs.
Forty-five dogs with severe respiratory signs caused by idiopathic, acquired laryngeal paralysis were treated by partial laryngectomy. The predominant postoperative complications were coughing in 28 dogs and pneumonia in 15 dogs. Eight dogs required a second operation to alleviate persistent or recurrent upper airway obstruction. Results of surgery were considered excellent in 11 dogs (25%), good in 18 dogs (40%), fair in 6 dogs (13%), and poor in 10 dogs (22%). Poorer results were obtained by surgical residents than by more experienced surgeons. Death in the immediate postoperative period was related to pneumonia (8 dogs) and laryngeal collapse (1 dog). Nine dogs died later of respiratory disease. Although partial laryngectomy is effective for the treatment of laryngeal paralysis, it is not recommended because of the high incidence of postoperative complications.
Airway Obstruction↗