Full Text (PDF)
The search for antibodies that can distinguish diseased cells from normal cells is of paramount importance. Serological identification of recombinantly expressed genes (SEREX), based on the detection of antigens within recombinantly expressed tumor cDNA phage libraries by autologous antibodies (1), has been used to isolate tumor or autoimmune antigens. SEREX combines serological analysis with antigen cloning techniques to identify human tumor antigens eliciting high-titer immunoglobulin G (IgG) antibodies (www.cancerimmunity.org/SEREX/introduction.htm) (2). This method has identified new tumor antigens that have subsequently been shown to contain epitopes for humoral immune responses (3,4). However, for efficient SEREX cloning, the serum titer against the antigen of interest needs to be high, the serum needs to be relatively specific, and large quantities of serum at the right point in time are necessary because the serum response may change over time (5). Furthermore, because B cells are present in the tissue used to construct the cDNA library, IgG cDNA may be incorporated into the library. Secondary anti-IgG antibodies could detect these protein fragments as false positives (5).
Typical phage display technology involves expression of recombinant proteins or peptides that are fused to a phage coat protein. Pertinent proteins, such as fused peptides, antibodies, and enzymes, may be synthesized and selected to acquire certain binding affinity and specificity (6,7,8,9). DNA fragments encoding millions of variants of protein fragments are cloned into the phage genome and expressed as a fusion protein as part of one of the phage coat proteins (10). Phage that display a certain protein ligand may be bound to an immobilized target and retained. Non-adherent phage are washed away, and the bound phage are recovered from the surface and subsequently used to re-infect bacteria for further enrichment.
To develop a system amenable to high-throughput to synthesize and produce purified recombinant antigens, we used agarose beads bound with an antibody to human SIRT2 as bait to screen a T7 phage human brain cDNA library. Human SIRT2 was used for these experiments since it is known to be expressed in brain. The protein is involved in the G2/M checkpoint in mitosis, and highly specific polyclonal antibody to SIRT2 was available (11). Four rounds of phage screening, referred to as biopanning, were found to produce an adequate number of reactive clones (approximately 8%). A problem with the use of phage lysates in immunological studies is that the majority of the proteins in the lysate are of host bacterial origin. Therefore, we developed a system in which the phage DNA in lysates were used as a template for a nested PCR that in turn generated a template for in vitro transcription/translation of the cloned epitope of interest. Utilizing the binding affinity of a His6-tag that was incorporated via the PCR steps, the recombinant protein was then purified by nickel affinity chromatography. This process can be performed in high-throughput to generate pure protein from the clones identified by phage display technology. Moreover, microarray chips containing purified proteins may be generated using clones that have been identified as reactive with disease versus normal human sera.
Functional analysis of crude and pure recombinant fusion protein was performed using peptide competition immunoassay on protein microarrays. The goal of this study was to develop a strategy for producing purified recombinant phage protein that can act as a superior antigen for future studies of serum antibodies as disease markers on protein microarrays. A flow chart of our technique is shown in (Figure 1).
Figure 1.
The isolation of a SIRT2 phage clone was achieved by differential biopanning technology. The first step in this strategy was to negatively immunoselect a T7 bacteriophage human brain cDNA library (Novagen, Madison, WI, USA) with pre-immune rabbit serum to remove the nontargeted proteins that react with pre-immune serum. The pre-adsorbed T7 phage library was then positively immunoselected with SIRT2 rabbit antisera, which acted as bait for selection of T7 bacteriophage bearing the SIRT2 insert. The biopanning procedure and phage propagation were done as per the manufacturer's instructions.
