Lutz Jermutus

Recent advances in producing and selecting functional proteins by using cell-free translation.

Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry. Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids. Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution. Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem.

Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.

Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment.

[24] Selecting and evolving functional proteins in vitro by ribosome display

Publisher Summary In vivo systems have a number of limitations. One important restriction is that the library size is limited by the transformation efficiency, a step that all of these techniques have in common. Another limitation of in vivo -based selection methods becomes apparent if a diversification step must be included. It requires either repeatedly switching between in vivo selection and in vitro diversification or the use of mutator strains. The former approach is quite laborious, as it makes a new library generation and large-scale transformation necessary for each cycle of sequence diversification and selection. The latter approach may have the disadvantage that possible candidate molecules may be removed from the library by mutations generated either in the host genome or in the plasmid regions important for expression or replication. All these limitations can be simultaneously overcome in ribosome display. Ribosome display is an in vitro technology for the simultaneous selection and evolution of proteins from diverse libraries. Because no transformation is necessary, large libraries can be prepared and applied for selection. Diversification is conveniently introduced in this method, thereby making evolutionary approaches easily accessible. Ribosome display was first applied to short peptides and has subsequently been improved to work with folded proteins. This chapter describes in detail the methodology and the nature of the improvements and provides the information necessary to perform ribosome display for the selection and evolution of functional proteins, using either an E. coli or a rabbit reticulocyte translation system.

Aggregation, stability, and formulation of human antibody therapeutics

Many human monoclonal antibodies display poor biophysical properties, such as low stability and a propensity to aggregate. These unfavorable tendencies can be even more pronounced for human antibody fragments, which often require a considerable degree of optimization. In this review, we describe methods for analyzing aggregation and stability of human antibodies and antibody fragments. We also provide an overview of recent approaches to improve these properties through engineering and formulation.

In vitro selection and evolution of proteins.

Publisher Summary This chapter discusses the in vitro translation, which is a prerequisite for devising and optimizing cell-free protein selection systems. In vitro protein synthesis, independent of its use in selection technology, this is due to the improvement of protein yields, the increase in detection sensitivity of many analytical methods, and the advent of new technologies, such as atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) that allow analytical work at low protein concentrations, even down to the single molecule level. Many applications of cell-free translation rely on the correct folding of the in vitro expressed polypeptide into its three-dimensional structure, and this is a prerequisite for all protein selection systems that are based on in vitro translation. Because proteins are selected and evolved for functionality, sufficient expression and correct folding in the respective cell-free translation system are a necessity for efficient selection. An attractive advantage of using in vitro translations is that, at least in principle, any component of the reaction can be deliberately added or removed. To achieve any improvement in yield, however, separate consideration of both the actual translation and the folding is necessary. Even in optimized systems, however, translation yields are not similar for all globular proteins.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

A human antibody facilitates opsonophagocytic killing, inhibits attachment of Pseudomonas aeruginosa, and exerts protective effects in several animal models of P. aeruginosa infection.

Probing a protein-protein interaction by in vitro evolution

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

Challenges and opportunities for non-antibody scaffold drugs.

The first candidates from the promising class of small non-antibody protein scaffolds are now moving into clinical development and practice. Challenges remain, and scaffolds will need to be further tailored toward applications where they provide real advantages over established therapeutics to succeed in a rapidly evolving drug development landscape.

Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems

Ribosome display is a technology for library selection and simultaneous molecular evolution in vitro. We present here a comparison between an optimized Escherichia coli system and different rabbit reticulocyte ribosome display systems, optimized in a number of parameters, as a coupled eukaryotic system had been suggested to result in high enrichment factors [He and Taussig (1997) Nucleic Acids Res. 25, 5132–5134]. With all systems, antibody scFv fragments, complexed to the ribosomes and the corresponding mRNA, were enriched by binding to their cognate antigen and enrichment was always dependent on the absence of a stop codon and the presence of cognate antigen. However, the efficiency of the E. coli ribosome display system was 100‐fold higher than an optimized uncoupled rabbit reticulocyte ribosome display system, with separate in vitro transcription and translation, which was in turn several‐fold more efficient than the reported coupled system. Neither the E. coli nor the rabbit reticulocyte ribosome display system was dependent on the orientation of the domains of an antibody scFv fragment or on the spacer sequence. In summary, we could not detect any intrinsic advantage of using a eukaryotic translation system for ribosome display.

Sustained peripheral depletion of amyloid-β with a novel form of neprilysin does not affect central levels of amyloid-β

Lowering levels of peripheral amyloid-β has been proposed as a strategy to reduce plaques in patients with Alzheimer’s disease. Henderson et al. test a modified version of the amyloid-degrading enzyme neprilysin in rats, monkeys and Tg2576 mice. Levels of amyloid-β were reduced in the bloodstream, but not in the CNS.