Production of recombinant human beta-catenin protein in Ecoli and screening for anti-beta-catenin

Abstract

The p-catenin gene is a component of wnt pathway involved in developmental pattern formation. The protein product is involved in both cell-cell interaction through E- cadherin and gene regulation through Tcf transcription fectors. In the absence of wnt signaling, P-catenin protein is degraded actively following phosphorylation of serine/threonine residues at its N-terminal region. In many cancer types (colorectal cancer, hepatocellular carcinoma, hepatoblastoma, melanoma, thyroid cancers...), P- catenin protein accumulates aberrantly due to a loss of active degradation process. In colorectal cancers, this is due to APC mutations, in others p-catenin mutations occur at or around exon 3 leading to a loss of degradation box of the protein product. It is believed that accumulated p-catenin protein result in increased cell growth by transcriptional activation of genes such as c-myc and cyclin D. A similar aberration was also observed with p53 gene whose mutations lead to the accumulation of mutant p53 proteins and loss of growth control. In addition, cancer patients expressing mutant p53 protein in their tumor cells develop anti-p53 autoantibodies, as well as cytotoxic T cell (CTL) response. Anti p-catenin CTL response in melanoma patients has been reported, but it is unknown whether cancer patients also develop anti-P-catenin autoantibodies. Our aim was to test whether cancer patients develop anti-P-catenin autoantibodies. For this aim, we first produced an N-terminally truncated recombinant P-catenin protein lacking the first 88 amino acid residues. The corresponding p-catenin cDNA was subcloned into an E. coli expression vector (pQE-32) with a bxHistidine tag at N-terminus. Affinity purified recombinant P-catenin protein was used as antigen to screen cancer patient and control sera by different immunoassays. Initially, more than 1500 sera were screened by an ELISA method and 53 sera were selected for further studies. The presence of anti-P-catenin autoantibodies was confirmed in 5 patients by western blot assay. All of these sera also reacted with in vitro produced full length catenin protein using immunoprécipitation assay. Five control sera were negative in this sensitive assay. This work demonstrated that some cancer patients develop autoantibodies to P-catenin. Such auto-antibodies may serve as tumor markers for patients displaying P-catenin mutations. Further studies are needed to determine the frequency and the specificity of anti p-catenin autoantibody response in different tumor types.