Transient and stable transfection of Chinese hamster ovary cells with the recombinant feline erythropoietin gene and expression, purification, and biological activity of feline erythropoietin protein.

OBJECTIVE: To use transient and stable transfection of Chinese hamster ovary cells to clone the gene encoding feline erythropoietin (feEPO) protein, characterize the expressed protein, and assess its biological activity. SAMPLE POPULATION: Cultures of Chinese hamster ovary or TF-1 cells. PROCEDURE: The gene encoding feEPO was cloned into a eukaryotic expression plasmid. Chinese hamster ovary cells were transiently or stably transfected with the plasmid. Expressed recombinant feEPO (rfeEPO) protein was purified from transiently transfected cells. The protein was characterized by use of SDS gel electrophoresis and western blot analysis. Biological activity was assessed by measuring thymidine incorporation by TF-1 erythroleukemic cells. RESULTS: Purified rfeEPO from supernatants of transiently transfected cells was determined to be 34 to 40 kilodaltons (kd) by use of SDS gel electrophoresis, whereas the molecular weight predicted from the amino acid sequence was 21.5 kd. The banding pattern and high molecular weight suggested the protein was glycosylated. The rfeEPO proteins derived from transient or stable transfections subsequently were determined to be biologically active in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: The gene encoding feEPO can be transfected into eukaryotic cells, and the expressed rfeEPO protein is biologically active in vitro. Cats with chronic renal failure often are anemic as a result of reduced expression of erythropoietin (EPO). Treatment with human-derived EPO stimulates RBCs in anemic cats; however, treatment is often limited by the development of antibodies directed against the recombinant human protein, which can then cross-react with endogenous feEPO. Recombinant feEPO may prove beneficial for use in cats with chronic renal failure.

Identification, characterization, and cloning of a complementary DNA encoding a 60-kd house dust mite allergen (Der f 18) for human beings and dogs.

BACKGROUND: House dust mites of the Dermatophagoides genus are the most important cause of perennial allergic disease in both humans and companion animals. Although the major mite allergens for humans are proteins of relatively low molecular weight, this is not the case for dogs. Western blotting shows that canine anti-mite IgE responses are directed primarily toward proteins in the molecular weight range of 50 to 120 kd. OBJECTIVE: The objectives of this study were to characterize a D farinae allergen with a molecular weight of approximately 60 kd and to isolate the cDNA coding for this allergen. METHODS: A protein of apparent molecular weight of 60 kd was identified by Western blotting by using canine serum IgE from house dust mite-sensitized atopic dogs. The protein was purified from homogenized D farinae mite bodies by ammonium sulfate precipitation, followed by gel filtration and cation exchange HPLC. The presence of IgE directed to the 60-kd protein in sera from humans and dogs with dust mite allergy was measured by FcepsilonRIalpha-based ELISA. A cDNA encoding a full-length 60-kd protein was isolated from a D farinae cDNA library by a combination of both PCR amplification and hybridization screening. A panel of mAbs specific for the 60-kd protein was generated and used to localize the protein in whole body sections of D farinae mites. RESULTS: ELISA showed that the purified protein bound IgE in 54% of the sera from patients with D farinae allergy. In addition, the 60-kd protein was able to bind IgE in 57% to 77% of D farinae -sensitized dogs. A cDNA was isolated that encoded a protein of 462 amino acids, consisting of a 25 amino acid signal sequence and a 437 amino acid mature protein. The calculated molecular weight of the mature protein is 50 kd, and the amino acid sequence contains a single N-glycosylation site. A protein database search showed homology with multiple chitinases. A mAb specific for the 60-kd chitinase recognized the allergen in the mite digestive system, but fecal pellets did not stain positively for this allergen. CONCLUSIONS: A 60-kd D farinae protein (Der f 18), with homology to chitinase, is a major allergen for humans and dogs sensitive to house dust mites.

Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis.

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.

Cloning, expression, purification, and biological activity of five feline type I interferons.

Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.