SubscribeFast and very efficient method for the generation of fully human antibodies against ESTs for protein expression profiling.
Genome projects have identified a huge number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products.
The availability of high-quality antibodies often is the limiting factor for carrying out functional studies on the increasing number of proteins discovered by these programs. To meet this demand, MorphoSys has devised a general method for the high-level expression of EST (or cDNA) encoded polypeptides, eligible for the high-throughput selection of antibodies.
Expressed Sequence Tags (ESTs) are gene fragments, which usually are the first evidence for the existence and expression of a gene. Although ESTs are generated in vast numbers as part of the Human Genome Project, the functions of the corresponding genes and gene products are mostly still unknown. Antibodies are first choice tools to determine the level of expression of the EST-encoded protein in various tissues of the organism, and to finally elucidate its function.
One approach is to express the ESTs and to generate antibodies by immunizing animals with the corresponding polypeptides. In a similar approach, DNA constructs comprising open reading frames have been injected into animals to generate an immune response against the polypeptide expressed in vivo. However, these approaches are not amenable to the high-throughput generation of antibodies.
Alternatively, antibodies can be generated against peptides derived from an EST or protein sequence. In combination with screening recombinant antibody libraries, this approach can in principle be developed to generate antibody fragments in a high-throughput mode.
However, it is difficult or even impossible to obtain anti-peptide antibodies with sufficiently high affinities. Moreover, to increase the probability of selection of binders recognizing the maternal protein, antibodies against different peptides derived from one target sequence have to be generated, leading to "target inflation".
Antibody generation against expressed protein fragments dramatically reduces screening efforts in comparison to peptide antigens. In addition, the resulting antibodies are more likely to bind the parental protein than antibodies against peptide fragments.
MorphoSys has developed a generally applicable method, called HuCAL® EST, for the high-throughput generation of antibodies to EST-encoded polypeptides. Expression of these polypeptides as fusion proteins in E. coli leads to high-level expression and formation of inclusion bodies.
After purification and refolding of these fusion proteins, the HuCAL® antibody library is panned for specific antibodies. These antibodies are then applied to IHC experiments for target research/validation purposes. The main advantages of this approach are (i) high-throughput, (ii) delivery of antibodies recognizing the maternal protein, (iii) high numbers of IHC-grade antibodies and (iv) good overall success rates.
We have chosen Escherichia coli as host, since expression of heterologous proteins in bacteria is by far the simplest and cheapest means to produce large amounts of the desired product. Simple expression of human genes or parts thereof, however, will not routinely lead to high yields, mainly due to inefficient translation initiation of eukaryotic mRNA in E. coli.
This limitation has been overcome by expression of recombinant eukaryotic protein sequences as a fusion with polypeptides that are well expressed and highly soluble in the cytoplasm of E.coli.
Soluble, heterologously expressed protein fragments will often not adopt an ordered, three-dimensional structure, and may become rapidly degraded in the cytoplasm of E. coli. Since we aimed at developing an expression method that can be generally applied in a robust, efficient mode, we genetically fused the EST or cDNA encoded protein fragments to the C-terminus of protein N1.
N1 is the N-terminal domain of g3p from filamentous phage M13, and offers the following advantages: (i) highly soluble and monomeric at millimolar concentrations, (ii) allows high-level expression of fusion proteins directed to inclusion bodies, thereby becoming protected against proteolysis and (iii) can easily be refolded, leading to soluble protein preparations.
We have demonstrated the success of this method for different fusion proteins containing fragments of various human proteins. Fusion proteins within a wide a range of molecular weights, and even those including a transmembrane domain, could be rapidly and efficiently expressed, purified, and refolded to yield soluble protein.
Refolding using a redox system offers the possibility of correct folding of proteins or domains having disulfide bonds, however we did not test the biological activity of the fusion proteins after the refolding process. The refolded protein preparation will probably consist of a mixture of functionally active, correctly folded domains and non-active, misfolded molecules.
Antibodies selected from pannings against EST-encoded polypeptide react either with the denatured, or the native maternal protein or with both. More than 40% of the selected antibodies detect the corresponding protein in IHC studies. Staining tissues with specific antibodies is a very powerful technology for target identification and validation for in situ protein expression profiling.
The high success rate of antibodies that recognize their maternal protein on Western Blot suggests that antibodies against misfolded molecules dominate. Since the fusion proteins are expressed in E. coli, one may not expect to generate antibodies against posttranslational modifications like glycosylations or phosphorylations.
Antibodies generated via HuCAL® EST are often the only way to determine the differential expression of an EST-encoded protein in multiple tissues. They support the determination of a protein's function and cellular location. (See Figures A and B, Protein Expression Analysis by IHC) Due to the fully human composition of these antibodies, they may immediately serve as lead candidates for therapeutic drug development.
HuCAL® antibodies against EST-encoded polypeptides have been successful in various immunoassays against fragments of various proteins from several species (e.g., human, mouse, rat, virus) and of different function and localization (e.g., receptors, integrins, hormons, transcription-factors).
The HuCAL® EST technology has proven to be a robust method for the high-throughput generation of large quantities of antigen suited for automated selection of antibodies for target research. To date, antibodies against approximately 100 different EST encoded polypeptides, derived mainly from target identification programs, have been generated using this technology.
Fab selected against EST / Negative control Fab
Figure B. Target for HuCAL® antibody selection: EST-encoded fragment (about 150 aa)
Murine intestine (formalin-fixed, paraffin-embedded)
Antibody format used: Fab_dHLX (dimeric Fab)
Fab selected against EST / Negative control Fab