There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10 10 individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.

No abstract

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.

Background:The EGF receptor (EGFR) is an important therapeutic target. Results: Bispecific anti-EGFR designed ankyrin repeat proteins (DARPins), alternative targeting molecules efficiently produced in bacteria, were shown to inhibit A431 cell proliferation and receptor recycling. Conclusion: One bispecific construct containing four DARPins showed a biological activity superior to that of the registered antibody cetuximab. Significance: Bispecific DARPins may form building blocks for tomorrow's cancer therapeutics.

This study examined the microbial dynamics associated with decomposing litter of the widespread emergent macrophyte Phragmites australis in a littoral reed stand of a large lake. Standing dead leaf and stem litter were collected, placed into fine and coarse mesh litter bags, and submerged in the reed stand. Litter bags were retrieved periodically and analyzed for fungal and bacterial biomass, fungal growth rates and production, rates of microbial respiration, litter mass loss, nutrient concentrations (N and P), and rates of dissolved organic carbon (DOC) release. Microbial biomass associated with both leaf and stem litter (12 to 85 mg C g -1 detrital C) was predominantly fungal (always ≥ 90% of the total biomass), even though bacterial biomass (0.13 to 5.6 mg C g -1 detrital C) increased and fungal biomass decreased or remained constant as litter decay proceeded. Although rates of fungal growth (0.02 to 0.08% h -1 ) and production (leaves only; 3 to 51 µg C g -1 detrital C h -1 ), and rates of microbial respiration (11 to 257 µg C g -1 detrital C h -1 ) decreased following litter submergence, fungi continued to be metabolically active in both leaf and stem litter. Significant differences in fungal and bacterial biomass, fungal production rates, and rates of respiration were observed between leaf and stem material, with leaves often having 5 times higher values than corresponding stems. Rates of mass loss differed significantly between leaf litter in fine and coarse mesh bags, with less than 10% of the initial mass remaining in coarse mesh bags after 86 d, versus nearly 60% remaining in fine mesh bags. Nitrogen and P concentrations of leaf litter enclosed in fine mesh bags increased during litter decay, whereas N concentrations of leaf litter in coarse mesh bags remained unchanged and P concentrations decreased. Both N and P concentrations of stem litter were similar among litter bags and varied little throughout the study period. Results obtained in this study indicate that significant changes in microbial colonization and activity associated with P. australis litter can occur following the collapse of standing dead plant matter to the water. Furthermore, these findings suggest that fungi are active on submerged litter and thus play a vital role in the decomposition of P. australis litter in the aquatic environment. KEY WORDS: Litter decomposition · Phragmites australis · Wetland · Microbial productivity · Fungi · Nutrients · Respiration · Growth efficiencyResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 22: 271-282, 2000 In many emergent macrophytes, such as Phragmites australis, abscission and collapse of plant material to the sediment or overlying surface waters typically do not occur immediately following shoot senescence and death. As a result, large amounts of dead plant matter remain standing within wetland habitats (Findlay et al. 1990, Lee 1990, Wetzel & Howe 1999, and are colonized and decomposed in an upright aerial position (Newell 1993, Ne...