A Cell-Free Protein Expression System Derived from Human Primary Peripheral Blood Mononuclear Cells

Abstract

Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.

Subcellular localization and relevant mechanisms of human cancer‐related micropeptides

“…Numerous experiments and techniques facilitate exploring endogenous and exogenous expression of micropeptides. Detecting exogenous expression primarily relies on designing vectors and transfecting target genes, immunofluorescence, immunoprecipitation, and cell‐free protein expression system 94 . Also, generating target antibody and CRISPR/Cas9 knock‐in TAG are used to detect endogenous expression 28 .…”

“…Detecting exogenous expression primarily relies on designing vectors and transfecting target genes, immunofluorescence, immunoprecipitation, and cell-free protein expression system. 94 Also, generating target antibody and CRISPR/Cas9 knock-in TAG are used to detect endogenous expression. 28 Given the continuous in-depth research, it is found that micropeptides function in transcription regulation, DDR, translation regulation, mitochondria fission, and so on.…”