3.1. NAPPA Technology for Understanding Proteins-Proteins Interactions
Four years after the first design of NAPPA technology, LaBaer’s group confirmed that protein function is maintained in printed proteins on high-density arrays. With this purpose, they designed an array expressing 647 unique genes in duplicate and tested for several well-characterized interactions, Jun-Fos and p53-MDM2 among others. Simultaneously, they expressed the corresponding protein printed on the array and co-expressed the query protein by adding the appropriate cDNA to the cell-free expression lysate. Using specific antibodies against Jun, Fos and MDM2, they detected specific interactions of these proteins. It is also necessary to take into account that protein function can be compromised by lack of PTMs and/or misfolding of certain domains due to the absence of chaperones and cofactors. Concerning the lack of PTMs, it is possible to use alternative cell free expression systems depending on the protein to be expressed. Thus, different expression systems have been developed (including HeLa, Leishmania, E. coli, rabbit expression systems, among others). Also, including ribosomal machinery and chaperones (such as HSP90 or HSC70) may encourage the folding of large multi-domain proteins [9].
More recently, in 2012, Fuentes and collaborators published a work in which a total of 450 mRNAs from O. moubata tick salivary glands were extracted and purified, and then transfected into a donor vector (pDONR222) generating a library of cDNA. Finally, this library was transfected again into a library destination expression vector (pANT7_GST), which allows in situ expression of GST-tagged proteins in cell-free systems. They built a NAPPA array randomly choosing 480 clones with validated sequences. After confirming successful display of the recombinant fused GST tag protein, the correct display of individual tick proteins was also checked with serum recognizing Om44, a P-selectin salivary protein from O. moubata whose neutralization induces antibody block tick feeding. To test the functionality of the proteins in the array, they performed protein-protein interaction studies with the recombinant P-selectin/Fc chimera. With this aim, the proteins on the array and P-selectin/Fc chimera were expressed in situ normally and also in the presence of canine pancreatic microsome membranes (CMMs). They found that P-selectin/Fc chimera interacted with phospholipase A2 (PLA2) expressed in situ on the array. This finding suggested that this secreted O. moubata PLA2 (sPLA2) could be a potential P-selectin interacting partner [11].
As another example, a NAPPA array was designed for systematic characterization of viral protein-host interactions. Through the access to viral ORFs in flexible cloning formats, the LaBaer’s lab is releasing the initiation of a panviral proteome collection of 2035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. In this work, NAPPA arrays are suitable, highly efficient and flexible platforms for displaying viral proteins and detecting host serological responses using micro-fluidic multiplexed immunoassays and allowing the study of host-viral protein interactions [12]. Related to host-pathogen interactions in Legionella pneumophila infections, this group have applied NAPPA technology to determine the interaction network of the pathogen with 10,000 unique human proteins. They identified known and novel interaction candidates and, additionally, substrates for an effector with and adenylyl transferase domain that catalyzes AMPylation. Their results highlighted the amenability of NAPPA to high-throughput analysis of effectors from a wide variety of human pathogens [13].
Nicolini and collaborators clinically screened neuro-oncological patients respondent to temozolomide (TMZ) from those showing resistance to the drug by using a NAPPA-based nanoconductometric sensor [14]. Their results shower a properly discrimination of protein-protein interactions depending on the behavior against TMZ [15]. Finally, Liang et al. have successfully coupled two different technologies (label-free and real-time detection method plasmonic-based electrochemical impedance microscopy with NAPPA arrays) to determine small molecule binding kinetics. This approach allowed the measurement of binding kinetics and affinity parameters between small molecule drugs (imatinib and SB201290) and their target proteins (kinases ABl1 and p38-α) with high sensitivity and reproducibility. These results demonstrate that NAPPA methodology is a reliable technology to understand small molecules interactions in biological systems and is also useful in the discovery of small molecules drugs [4].