Self-assembling functional programmable protein array for studying protein–protein interactions in malaria parasites

Abstract

BackgroundPlasmodium vivax is the most widespread malarial species, causing significant morbidity worldwide. Knowledge is limited regarding the molecular mechanism of invasion due to the lack of a continuous in vitro culture system for these species. Since protein–protein and host–cell interactions play an essential role in the microorganism’s invasion and replication, elucidating protein function during invasion is critical when developing more effective control methods. Nucleic acid programmable protein array (NAPPA) has thus become a suitable technology for studying protein–protein and host–protein interactions since producing proteins through the in vitro transcription/translation (IVTT) method overcomes most of the drawbacks encountered to date, such as heterologous protein production, stability and purification.ResultsTwenty P. vivax proteins on merozoite surface or in secretory organelles were selected and successfully cloned using gateway technology. Most constructs were displayed in the array expressed in situ, using the IVTT method. The Pv12 protein was used as bait for evaluating array functionality and co-expressed with P. vivax cDNA display in the array. It was found that Pv12 interacted with Pv41 (as previously described), as well as PvMSP142kDa, PvRBP1a, PvMSP8 and PvRAP1.ConclusionsNAPPA is a high-performance technique enabling co-expression of bait and query in situ, thereby enabling interactions to be analysed rapidly and reproducibly. It offers a fresh alternative for studying protein–protein and ligand–receptor interactions regarding a parasite which is difficult to cultivate (i.e. P. vivax).Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2414-2) contains supplementary material, which is available to authorized users.

Lithographic Processes for the Scalable Fabrication of Micro- and Nanostructures for Biochips and Biosensors

“…From the perspective of their fabrication, in addition to the considerations that apply to oligonucleotide chips discussed above, the process involved must be able to maintain the correct protein folding and hence their biochemical function . A widely adopted method for the production of functional protein microarrays involves in situ transcription and translation of DNA arrays, which was first reported in 2004 by LaBaer et al and termed “nucleic acid programmable protein arrays” (NAPPA). , In NAPPA, the translated proteins are captured under physiological conditions (see following section), and the translation is carried out only immediately prior to analysis, thus avoiding the loss of protein integrity or activity, while offering reproducibility and throughput. − Other approaches involve the deposition onto the chip surface of short peptides or proteins produced from standard methods (see following sections).…”

Plasmodium 6-Cysteine Proteins: Functional Diversity, Transmission-Blocking Antibodies and Structural Scaffolds

“…A panel of 20 P . vivax proteins including Pv12 were expressed by nucleic acid programmable protein array/ in vitro transcription/translation, confirming the interaction of Pv12 with Pv41 as well as identifying additional putative interaction partners ( Arevalo-Pinzon et al., 2018 ).…”

SARS-CoV-2 Infection Triggers Auto-Immune Response in ARDS

“…Based on previous reports (22), NAPPA was built with cDNAs encoding ARDS AABs in triplicate and positive (Cy3, MasterMix) and negative (GST-antibody, PBS, bovine serum albumin (BSA), Bis-(sulfosuccinimidyl) suberate (BS3), clean buffer, and printing buffer) controls (Table S6). The design and distribution of the cDNAs on the arrays was as follows: 6 subarrays, each subarray with 144 spots, deposited on a chemically activated surface prepared accordingly with ArrayJet Printer Marathon v1.4.…”