Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.     

Nicotine's Defensive Function in Nature

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D₄-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Variation in number and differentiation of the abdominal infrared receptors in the Australian 'fire-beetle' Merimna atrata (Coleoptera, Buprestidae)

Most individuals of the Australian ‘fire-beetle’ Merimna atrata have two pairs of IR receptors which are located ventrolaterally on the second and third abdominal sternite. An IR receptor consists of a specialized IR absorbing area, which is innervated by a neural complex. This complex contains one thermoreceptive multipolar neuron with a unique terminal dendritic mass (TDM) and two scolopidia and was termed ‘sensory complex’. However, also individuals with one pair of IR receptors on the second sternite and beetles with three pairs on the second, third, and fourth sternites were found. Additionally, beetles having one or two pairs of IR receptors may have preliminary stages of IR receptors on the third and fourth sternite, respectively. We found two kinds of preliminary stages, both of which are characterized by a much less pronounced absorbing area. In all five abdominal sternites segmental nerves are attached to the cuticle with a neural complex. Investigation of complexes of non-IR sternites suggests that the sensory cells inside the sensory complex of an IR receptor have developed from common internal stretch receptors. From our results it can be hypothesized that the IR sensory system in Merimna atrata has not yet reached a stage, which can be regarded as evolutionary stable.     

A single intratracheal dose of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid differential increase of dendritic cells and lymphocyte subsets in lung tissue and bronchoalveolar lavage,resulting in an increased local antibody production

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4(+) and CD8(+)), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.     

Development of resistance against diketo derivatives of human immunodeficiency virus type 1 by progressive accumulation of integrase mutations

The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer step of the human immunodeficiency virus type 1 (HIV-1) integration process (D. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller, Science 287:646-650, 2000). We have now studied the development of antiviral resistance to L-708,906 by growing HIV-1 strains in the presence of increasing concentrations of the compound. The mutations T66I, L74M, and S230R emerged successively in the integrase gene. The virus with three mutations (T66I L74M S230R) was 10-fold less susceptible to L-708,906, while displaying the sensitivity of the wild-type virus to inhibitors of the RT or PRO or viral entry process. Chimeric HIV-1 strains containing the mutant integrase genes displayed the same resistance profile as the in vitro-selected strains, corroborating the impact of the reported mutations on the resistance phenotype. Phenotypic cross-resistance to S-1360, a diketo analogue in clinical trials, was observed for all strains. Interestingly, the diketo acid-resistant strain remained fully sensitive to V-165, a novel integrase inhibitor (C. Pannecouque, W. Pluymers, B. Van Maele, V. Tetz, P. Cherepanov, E. De Clercq, M. Witvrouw, and Z. Debyser, Curr. Biol. 12:1169-1177, 2002). Antiviral resistance was also studied at the level of recombinant integrase. Single mutations did not appear to impair specific enzymatic activity. However, 3' processing and strand transfer activities of the recombinant integrases with two (T66I L74M) and three (T66I L74M S230R) mutations were notably lower than those of the wild-type integrase. Although the virus with three mutations was resistant to inhibition by diketo acids, the sensitivity of the corresponding enzyme to L-708,906 or S-1360 was reduced only two- to threefold. As to the replication kinetics of the selected strains, the replication fitness for all strains was lower than that of the wild-type HIV-1 strain.     

Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.