CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins

Abstract

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.

Development of a cell-free protein synthesis system for practical use

“…With this technology, a human protein library has been constructed. 67 , 68 ) The Protemist DTII is equipped with functions for transcription, translation, and affinity purification of up to 6 samples in a medium reaction size of 6 ml (Fig. 7 B).…”

CF-PPiD technology based on cell-free protein array and proximity biotinylation enzyme for in vitro direct interactome analysis

“…In the antibody validation system, CF-PA 2 Vtech, the human recombinant proteins for protein array were synthesized as FLAG-GST fusion proteins (FG-fusion proteins) 21 . In the same study, a magnetic plate device was developed to capture these proteins by glutathione-conjugated magnetic beads via an N -terminal fused GST protein.…”

“…Human protein arrays have been reported by several groups, including our group 20 , 21 . In our previous study, a human protein array consisting of 19,712 recombinant proteins was constructed by the combination of a wheat cell-free protein production system and a full-length human cDNA set 21 . Multiple-well plate or glass-slide formats were mainly used as the screening devices for the protein array 22 , 23 .…”

“…Multiple-well plate or glass-slide formats were mainly used as the screening devices for the protein array 22 , 23 . Recently, we have developed a strong magnetic plate for capturing recombinant proteins and showed that the plate can be used for the validation of antibody specificity 21 . In the present study, we used this magnetic plate to develop a biochemical screening system for PPI analysis by combining BioID technology with a human protein array, which we termed cell-free human protein array technology and proximity biotinylation-based PPI identification (CF-PPiD).…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…Moreover, the in vitro reaction format of CFPS systems allows for full automation, miniaturization ( Ayoubi-Joshaghani et al, 2020 ), and working with large sample numbers ( Zhu et al, 2015 ). This advantage has been utilized in large-scale screening experiments ( Khnouf et al, 2010 ; Kim et al, 2015 ), searches for malaria vaccine candidates ( Kanoi et al, 2017 ; Morita et al, 2017 ; Kanoi et al, 2020 ), identifying interactions between E3 ligases and their substrates ( Takahashi et al, 2016 ), building a protein array holding human Deubiquitinating Enzymes (DUBs) ( Takahashi et al, 2020 ), or the development of protein array platforms ( Romanov et al, 2014 ; Zarate and Galbraith, 2014 ; Morishita et al, 2019 ). Other promising developments make use of the stability of the reagents, where the extracts and buffers can be lyophilized for long-term storage at room temperature ( Smith et al, 2014 ).…”