A human scFv antibody generation pipeline for proteome research.

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.

Highly efficient gene targeting in the Aspergillus niger kusA mutant.

Gene targeting frequencies in Aspergillus niger are often very low and hamper efficient functional genomics in this biotechnologically important fungus. Deletion of the A. niger kusA gene encoding the ortholog of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency and reached more than 80% compared to 7% in the wild-type background, when 500bp homologous flanks were used. Furthermore, the use of the DeltakusA strain resulted in a high frequency of heterokaryon formation (70%) in primary transformants in the case disrupting an essential gene. Deletion of kusA had no obvious effect on the growth of the fungus, but renders the DeltakusA strain 10 times more sensitive to X-ray irradiation and two to three times more sensitive to UV exposure. The highly efficient gene targeting in combination with the A. niger genome sequence allows a systematic approach to generate gene knockouts and will help in improving the capacities of A. niger as producer of commercially interesting proteins and metabolites.

Oceanibulbus indolifex gen. nov., sp. nov., a North Sea alphaproteobacterium that produces bioactive metabolites.

A water sample from the North Sea was used to isolate the abundant heterotrophic bacteria that are able to grow on complex marine media. Isolation was by serial dilution and spread plating. Phylogenetic analysis of nearly complete 16S rRNA gene sequences revealed that one of the strains, HEL-45T, had 97.4% sequence similarity to Sulfitobacter mediterraneus and 96.5 % sequence similarity to Staleya guttiformis. Strain HEL-45T is a Gram-negative, non-motile rod and obligate aerobe and requires sodium and 1-7% sea salts for growth. It contains storage granules and does not produce bacteriochlorophyll. Optimal growth temperatures are 25-30 degrees C. The DNA base composition (G+C content) is 60.1 mol%. Strain HEL-45T has Q10 as the dominant respiratory quinone. The major polar lipids are phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine and an aminolipid. The fatty acids comprise 18 : 1omega7c, 18 : 0, 16 : 1omega7c, 16 : 0, 3-OH 10 : 0, 3-OH 12 : 1 (or 3-oxo 12 : 0) and traces of an 18 : 2 fatty acid. Among the hydroxylated fatty acids only 3-OH 12 : 1 (or 3-oxo 12 : 0) appears to be amide linked, whereas 3-OH 10 : 0 appears to be ester linked. The minor fatty acid components (between 1 and 7%) allow three subgroups to be distinguished in the Sulfitobacter/Staleya clade, placing HEL-45T into a separate lineage characterized by the presence of 3-OH 12 : 1 (or 3-oxo 12 : 0) and both ester- and amide-linked 16 : 1omega7c phospholipids. HEL-45T produces indole and derivatives thereof, several cyclic dipeptides and thryptanthrin. Phylogenetic analysis of 16S rRNA gene sequences and chemotaxonomic data support the description of a new genus and species, to include Oceanibulbus indolifex gen. nov., sp. nov., with the type strain HEL-45T (=DSM 14862T=NCIMB 13983T).