*2.1.2. Functional protein arrays or recombinant protein arrays*

It is based in the identification of protein interactions with different molecules (proteins, DNA, lipids, drug, etc.), so recombinant proteins are printed onto array surface.

Some examples are protein in situ array (PISA), printing arrays from DNA (DAPA) nucleic acid programmable protein array (NAPPA) and multiplexed nucleic acid programmable protein array (M-NAPPA). They are essential for pharmaceutical industry because they allow to know protein interactions [11].

PISA is based on DNA amplified by PCR as a template. The DNA that encode the protein of interest contains a T7 promoter or another strong transcriptional promoter and an in-frame N-12 or C-terminal tag sequence for protein capture onto the surface. PISA offers the possibility to cell-free production of protein arrays. It means protein are produced using cell extracts directly on the surface os arrays. PISA demonstrated that multiple proteins could be produced without the need of using cells for expression followed by lysis and purification to make the proteins [11].

DAPA is a technique derived from PISA, but DAPA allows use the same DNA template slide repeatedly for printing up to 20 copies of the same protein and also DNA could be reused after prolonged periods of time. DAPA takes a long time to express proteins due to the diffusion of proteins through the membrane. This technique starts by spotting the PCR amplified DNA fragments encoding the tagged protein on one slide. This slide is sandwiched with another Ni-NTA slide where a tag-capturing agent immobilizes the expressed protein. A permeable membrane with the cell-free lysate which allows coupled transcription and translation is places between the two slides. Then, the expressed proteins are captured to the surface on the other slide through the capture ligand. Overall, DAPPA requires long time to express proteins and this technique presents the protein diffusion as strong limitation, in particular with large proteins [7, 9, 11, 12].

NAPPA uses cDNA templates cloned into expression plasmids which adds a transcriptional promoter and also an in-frame polypeptide capture tag. It has several advantages:- (1) once the clone is produced as a glycerol stock it becomes an indefinitely renewable- resource that could be shared with other labs; (2) if the clone is carefully sequence verified,- then the resource will have long-term sequence fidelity; (3) the use of plasmids removes- some of the length constraints on the epitope tags, so that functional protein tags can be used. There are many applications of NAPPA where the proteins are fused with glutathione-S-transferase (GST); nevertheless, other tags such as flag, HA, c-myc, and Halo- tag have been used in specific applications. High quality supercoiled plasmid DNA is- purified from bacteria cultures and printed onto an activated ester surface along with a- homo-bifunctional crosslinker, bovine serum albumin (BSA) and anti-GST antibody. BSA efficiently increased the DNA binding and narrows down the unspecific interactions and- anti-GST attaches the protein expressed. When cell-free expression system is added to the- array, a coupled transcription/translation reaction is produced and the nascent protein is linked to the capture agent tag the C-terminal end assuring the complete translation of the- protein [10, 11, 13].

Puromycin capture protein arrays (PuCA) are cell-free expression protein arrays based on the affinity of puromycin by expressed peptide/protein. First, PCR DNA is transcribed to mRNA, and a single-stranded DNA oligonucleotide modified with biotin and puromycin on each end is then hybridized to the 3′-end of the mRNA.-The mRNAs are placed on a slide and immobilized by the binding of biotin to streptavidin which is previously coated on the slide. Cell extract is then dispensed on the slide for in situ translation to take place. When the ribosome reaches the hybridized oligonucleotide, it stops and incorporates the puromycin molecule to the nascent polypeptide chain, thereby attaching the newly synthesized protein to the microarray through the DNA oligonucleotide [14].

M-NAPPA is based on combining up to five different DNA plasmids at one point. This increases the number of proteins displayed by microarrays by five. It also reduces the cost and the time spent in work. M-NAPPA would be useful in unbiased HT screening studies, such as protein-protein interactions, protein-DNA interactions, discovery of drug binding target as well as (auto)antibody biomarkers for a variety of human diseases [15].
