Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Cleavage of beta-carotene as the first step in sexual hormone synthesis in zygomycetes is mediated by a trisporic acid regulated beta-carotene oxygenase.

Carotene cleavage is the necessary initial step in the biosynthesis of trisporic acid, the sexual signal in zygomycete fungi. Two genes encoding putative carotene oxygenases, designated tsp3 and tsp4, were identified in the genome of the zygomycete Rhizopus oryzae. Using heterologous primers, tsp3 was cloned and sequenced also from Blakeslea trispora. tsp3 transcription correlates with sexual development in both species. Northern hybridization of B. trispora mRNA revealed strong induction of tsp3 transcription in mated cultures. A very strong and direct transient induction of transcription by trisporic acid was proven by quantitative real-time PCR analysis. In R. oryzae, transcriptional induction is also inducible by stimulation with trisporoids and depends on the developmental stage of the mycelium. The functionality of the tsp3 gene product as carotene cleavage enzyme was shown as loss of carotene in an Escherichia coli strain transformed to carotene production and tsp3 expression.

4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types.

4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.

Sexuality and parasitism share common regulatory pathways in the fungus Parasitella parasitica.

Parasitella parasitica, a facultative mycoparasite of zygomycetous fungi, forms cytoplasmic fusions with its hosts during infection. Thus, the organism is an efficient donor of genetic material in parasexual host-parasite interactions. Recognition between parasite and host is mediated by trisporoids, which are also responsible for sexual communication. The TDH gene for one of the key enzymes of trisporic acid biosynthesis, 4-dihydromethyl-trisporate dehydrogenase, was cloned and its transcription analysed. TDH was cloned on a 6175-bp insert and was found to map in a complex cluster of genes that suggest post-transcriptional antisense regulation. Histochemical TDH analysis in developing parasitic or sexual structures shows high enzymatic activity in Parasitella. TDH is linked to a gene for a putative acyl-CoA thioesterase (ACT). Two ORFs were identified in the 5'-region of the TDH gene, a third one, coding for 176 amino acids overlaps the ACT gene in antisense direction completely. Expression levels of ACT and ORF1 depend on parasitic and sexual interactions.