Automated unrestricted multigene recombineering for multiprotein complex production

Abstract

Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…The ACEMBL technology was conceptualized to meet these requirements, to enable structural and functional "complexomics" research and discovery [31][32][33][34]. ACEMBL comprises recombination-based assembly of DNA elements into functional multigene expression constructs that can be rapidly permutated in a combinatorial fashion [33].…”

“…ACEMBL comprises recombination-based assembly of DNA elements into functional multigene expression constructs that can be rapidly permutated in a combinatorial fashion [33].…”

“…Originally, ACEMBL was developed for combinatorial multiprotein production in E.coli as a prokaryotic expression host [33]. Subsequently, efficient ACEMBL tool-kits have been developed also for multigene expression in eukaryotic hosts [34][35][36][37].…”

“…Recombinant production offers solutions to these impediments, and a wide range of expression systems are available to produce proteins recombinantly in prokaryotic and eukaryotic hosts [31,33,[34][35][36][37][38][39][40][41][42][43][44][45]. Recombinant expression systems share in common that one or several DNA segments encoding for proteins, protein domains or multicomponent protein complexes are typically combined with DNA elements including DNAs that control transcription (promoters, terminators, others) and translation (ribosome binding sites, ShineDalgarno sequences, Kozak consensus sequences, enhancers, others) and inserted into a functional DNA module (plasmid, cosmid, artificial chromosome, genome, others).…”

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

“…The rising of protein-protein interaction studies as well as the constantly increasing demand of proteins in drug discovery programs have brought the need for high-throughput protein expression and automation to the foreground [1][2][3][4][5]. The need for recombinant proteins with post-translational modifications has fostered the development of mammalian cell-based expression systems rather than prokaryotes and lower eukaryotes [6].…”