Immunohistology

Figure 7. A flow chart representation showing how the dual expression concept has been used in functional cDNA research. The concept is thus used for the generation of highly specific antibodies employed in various analyses to assign function to cDNA-encoded proteins. Such sera have indeed shown to be useful to localize gene products to a certain cell type within a tissue section and also further down to subcellular levels (13).

highly enriched antisera should be suitable for functional analysis of the cDNA-encoded protein, for example, in Western blots and immunohistology on tissue sections or within cells. The principle was evaluated by expressing five cDNAs, isolated from prepubertal mouse testis by a differential cDNA library screening strategy. All five clones could be expressed intracellularly in E. coli as fusion proteins with high production levels, and affinity purification yielded essentially full-length products. Characterization of affinity-purified antibodies revealed that there existed no cross-reactivity between the two fusion systems, and that such antibodies indeed were valuable for various analyses to elucidate the functions of the cDNA-encoded proteins (13). For example, in the presented study a previously unknown cDNA, with no sequence homology to genes in the public databases, could be connected with a gene product found in nuclear bodies within spermatogonial cells (13).

To make it possible to use the dual expression system for functional analysis of cDNA, where robustness of the expression systems is crucial, completely new expression vectors were constructed. Intracellular expression was used and transcription was tightly controlled by the T7 promoter system (13). This method should ensure that a higher fraction of investigated cDNA-encoded proteins are successfully produced. The different ongoing genome projects have generated enormous amounts of partially se-quenced cDNAs, and there exists a significant need for various alternative systems to take care of this informa tion to elucidate the function of the corresponding proteins. By automation of some of the unit operations, the system described by Larsson and coworkers (13) might have the potential to make large-scale functional analysis of cDNA libraries possible.

Bacterial Surface Display

The display of heterologous proteins on the outer surface of bacteria has become an emerging topic in different fields of research within biotechnology, microbiology, and immunology (125,126). Genetic insertion of a target sequence into the genes for various outer membrane proteins has constituted the general strategy to enable secretion and subsequent surface anchoring of the recombinant target gene products. For Gram-negative bacteria, represented by E. coli and Salmonella spp., a number of different types of heterologous proteins have been surface displayed including antigenic determinants for the purpose of vaccine development, enzymes, metal-chelating histidyl peptides, and single-chain antibodies, as well as entire peptide libraries (125-128). Recently, surface display on Grampositive bacteria has gained interest in the context of vaccine development. Approaches based on the attenuated mycobacteria (129), commensal streptococci (130), and nonpathogenic food-fermenting staphylococci have been presented (48,76-78).

To achieve surface display of various target proteins, the staphylococcal systems take advantage of the cell surface anchoring regions (XM) of SpA (see Fig. 1). The surface-exposed proteins are thus genetically fused between a functional signal peptide, responsible for the translocation from the cell, and the XM-regions of SpA. Heterologous antigenic determinants of various origin (bacteria, protozoa, and human virus) have been expressed as anchored on recombinant staphylococci, which are being investigated as a bacterial vaccine delivery system (48,7678,80,131-133). Even active single-chain antibody fragments have been surface displayed on recombinant staph-ylococci (79). Such recombinant bacteria could be envisioned as inexpensive tools in diagnostic tests.

Protein A Domains and Phage Technology for Selection of "Affibodies"

An example of emerging interest in which gene fusions have made new technology possible is the display of libraries of peptides or proteins on filamentous phage surfaces. Phage particles decorated in such manner can be enriched in vitro from a background of irrelevant phages by biopanning techniques, employing an immobilized li-gand capable of binding to the displayed protein. Moreover, inside its protein coat the captured phage carries the genetic information of the displayed protein, which allows later identification by DNA sequencing. Thus, the technology provides a link between genotype and phenotype and allows the selection of peptides or proteins of desired functions to be expressed on the surface of the filamentous phage. This technology, as pioneered by Smith (134), has emerged into a powerful technology to select, rather than screen, for rare phages capable of binding to a defined target molecule from libraries comprising as many as 1010

phage species. Such phage libraries are obtained by the fusion to phage pIII or pVIII genes, of cassettes encoding randomized peptides (usually 6- to 10-mers), antibody repertoires, or multiple variants of a protein (135). Thus, it is possible to rapidly and efficiently select high-affinity li-gands or effector molecules that can be used for analytical or preparative applications.

The Z-domain from SpA has several features including proteolytic stability, high solubility, small size, and compact and robust structure, which makes it ideal as a fusion partner for the production of recombinant proteins. The residues responsible for the IgG Fc-binding activity have been identified from the crystal structure of the complex between its parent B-domain and human IgG Fc, showing that these residues are situated on the outer surfaces of two of the helices making up the domain structure and do not contribute to the packing of the core (37). The Fc-binding surface covers an area of approximately 600 A2, similar in size to the surfaces involved in many antigen-antibody interactions. Taken together, these data suggest that if random mutagenesis of this binding surface was performed, novel domains could be obtained with the pos sibility of finding variants capable of binding molecules other than Fc of IgG. In addition, these domains would theoretically share the overall stability and a-helicalstruc-ture of the wild-type domain.

Recently, an attempt to obtain such ligands with completely new affinities was initiated, employing phage display technology (136) (Fig. 8a). A genetic library was created in which 13 surface residues (involved in the IgG Fc binding) of the Z-domain were randomly and simultaneously substituted to any of the 20 possible amino acids. This Z-library has, after genetic fusion to the gene for coat protein 3 of filamentous phage M13, resulted in a phage library adapted for selection of novel specificities by bio-panning (137). The library has been subjected to such affinity selection against different molecules for investigation as a source of novel binders (Fig. 8b). So far, novel Z-variants, so-called affibodies, have successfully been selected to diverse targets, such as Taq DNA polymerase, human insulin, and a human apolipoprotein variant (137). This concept suggests that the described strategy might prove to be beneficial in generating a "next generation" of ligands or artificial antibodies to various target molecules,

M13 filamentous phage

Fc binding surface = target area for randomization

Helix 1

Fc binding surface = target area for randomization

Helix 1

Helix 2

Hydrophobic core

Helix 2

Novel molecular surface, mediating binding to desired target molecule

1. Construction of phage-displayed library

2. Selection using biopanning

3. Isolation of domains with novel specificities

Hydrophobic core

Novel molecular surface, mediating binding to desired target molecule

Helix 3 Wild-type Z-domain

Variants with novel binding specificities

Figure 8. Schematic description showing how the Z-domain has been used as molecular scaffold in search of domain variants with novel binding specificities. (a) Using phage display technology, Z-variants can be expressed for presentation on the surface of filamentous phage particles. Phage libraries containing large numbers of phage clones, each displaying an individual Z-variant, can be subjected to rounds of biopannings in which Z-variants capable of binding to a desired target molecule can be selected. Identification of binding variants is accomplished through DNA sequencing of the packed phage DNA, containing the gene of the displayed Z-variant. (b) Top view of the Z-domain consisting of a triple a-helix. The library is constructed through randomization of amino acids present at the domain surface and involved in the Fc binding of the wild-type domain. Using the selection procedure (biopanning), variants with new molecular surfaces are isolated that are capable of binding the desired target.

to be used for purification, detection, immobilization, and perhaps even therapeutic purposes.

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