Multiple factors are involved in the translation of functional genomics results into proteins for proteome research and target validation on tumoral tissues. In this report, genes were selected by using DNA microarrays on a panel of colorectal cancer (CRC) paired samples. A large number of up-regulated genes in colorectal cancer patients were investigated for cellular location and those corresponding to membrane or extracellular proteins were used for a non-biased expression in E.coli. We investigated different sources of cDNA clones for protein expression, as well as the influence of the protein size and the different tags with respect to protein expression levels and solubility in E. coli. From 29 selected genes, 21 distinct proteins were finally expressed soluble with, at least, one different fusion protein. In addition, seven of these potential markers (ANXA3, BMP4, LCN2, SPARC, SPP1, MMP7 and MMP11) were tested for antibody production and/or validation. Six of the seven proteins (all except SPP1) were confirmed to be overexpressed in colorectal tumoral tissues by using immunoblotting and tissue microarray analysis. Although none of them could be associated to early stages of the tumour, two of them (LCN2 and MMP11) were clearly overexpressed in late Dukes stages (B and C). This proteomic study reveals novel clues for the assembly of a robust and highly efficient high throughput system for the validation of genomic data. Moreover, it illustrates the different difficulties and bottlenecks encountered for performing a quick conversion of genomic results into clinically useful proteins.