Application of immobilized bovine enterokinase in repetitive fusion protein cleavage for the production of mucin 1

Bovine enterokinase is a serine protease that catalyzes the hydrolysis of peptide bonds and plays a key role in mammalian metabolism. Because of its high specificity towards the amino acid sequence (Asp)₄-Lys, enterokinase is a potential tool for the cleavage of fusion proteins, which are gaining more importance in biopharmaceutical production. A candidate for adaptive cancer immunotherapy is mucin 1, which is produced recombinantly as a fusion protein in CHO cells. Here, we present the first repetitive application of immobilized enterokinase for the cleavage of the mucin fusion protein. The immobilization enables a facile biocatalytic process due to simplified separation of the biocatalyst and the target protein. Immobilized enterokinase was applied in a maximum of 18 repetitive reactions. The enzyme utilization (total turnover number) was increased significantly 419-fold compared to unbound enzyme by both immobilization and optimization of process conditions. Slight enzyme inactivation throughout the reaction cycles was observed, but was compensated by adjusting the process time accordingly. Thus, complete fusion protein cleavage was achieved. Furthermore, we obtained isolated mucin 1 with a purity of more than 90% by applying a simple and efficient purification process. The presented results demonstrate enterokinase to be an attractive tool for fusion protein cleavage.     

Zygotic Lethal Mutations with Maternal Effect Phenotypes in Drosophila Melanogaster. II. Loci on the Second and Third Chromosomes Identified by P-Element-Induced Mutations

Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal ``FLP-DFS' technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.     

The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death

The segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.     

Studies on proteins of animal ribosomes

Summary Ribosomes from different tissues and species of animals were tested by several different immunochemical methods. With antisera produced in rabbits by injection of intact ribosomes significant species differences in the antigenic properties of the ribosomes could be demonstrated whereas no tissue conditioned properties in the antigenic determinants were found. Abbreviations. RRL: ribosomes of rat liver; RBL: ribosomes of bovine liver; RBK: ribosomes of bovine kidney; anti-RRL: antiserum against RRL; anti-RBL: antiserum against RBL; anti-RBK: antiserum against RBK.     

The role of physiological oxygen partial pressures in lipid peroxidation. Theoretical considerations and experimental evidence

The oxidative breakdown of membrane lipids, i.e. lipid peroxidation, is considered to be decisively involved in a number of toxicological and pathological processes including liver injury caused by iron overload and halogenated alkanes such as CCl4. Within the physiological oxygen partial pressure (PO2) range, i.e. at PO2 between 1 and 100 mmHg, lipid peroxidation depends on O2 in a complex manner. For instance, CCl4-induced lipid peroxidation exhibits a distinct maximum at PO2 of around 7 mmHg, and iron-induced lipid peroxidation shows marked differences in its O2 dependence between an early lag phase and a later phase of self-accelerating propagation. The O2 dependence of lipid peroxidation is either determined by the O2 dependence of initiation or the O2 dependence of propagation. Factors decisive for this are presented and the underlying alterations in the pattern of the peroxidation-related reactions delineated.     

Bile acids. 38. Conversion of 5 -cholestane-3 ,7 -diol to allo bile acids by the rat

5alpha-[4-(14)C, 3alpha-(3)H]Cholestane-3beta,7alpha-diol was prepared from individual samples of 5alpha-[3alpha-(3)H]cholestane-3beta,7alpha-diol and 5alpha-[4-(14)C]cholestane-3beta,7alpha-diol, each derived from 3beta-acetoxycholest-5-en-7-one. Bile was collected for 11 days from adult male rats, with cannulated bile ducts, that had received intraperitoneally 0.90-0.92 mg of the doubly labeled diol. Bile from the first 10 hr, containing 63% of the administered (14)C and 6% of the (3)H, was hydrolyzed, and the bile acids were separated by acetic acid partition chromatography. Allochenodeoxycholic and allocholic acids contained at least 20.6% and 48.6%, respectively, of the (14)C retained in the biliary acids. Small amounts of (14)C (2.5% and 1.9%, respectively) were present in the 3beta isomers of these acids, but the tritium content totaled more than half of that found in the bile acid fraction. No evidence was obtained for presence of the extensive quantities of the allomuricholates.