Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins

Abstract

Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins, mAbs, 6:3, 671-678,

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…Extract SBDG108 was chosen because it expresses both DsbC and FkpA, 2 chaperons that are essential for IgG folding and assembly. 48 Small scale reactions at 100 mL were performed in a 96-well microtiter plate with a breathable cover while shaking at 650 rpm for 14 hours in an Eppendorf Thermomixer R. For protein production purposes, larger scale reactions (20-50 mL) were set up in a thinfilm petri dish format without shaking.…”

“…46,47 Without the limitation of cell viability imposed by conventional expression systems, Xpress CF is capable of protein expression at g/L scale within hours, and has been demonstrated to retain high productivity in transition from micro-to manufacturing scale. Its open and flexible nature can tolerate multifarious manipulations in extract, DNA template, and chemical and protein additives, 46,48,49 which makes it amenable for high-throughput screening by automation. 50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins.…”

Cell-Free Synthesis Meets Antibody Production: A Review

“…In a very recent report, Groff and colleagues published a potent cell-free translation system based on a chaperoneenriched E. coli cell extract enabling the synthesis of full length IgG molecules in gram per liter quantities. In this study, the authors were able to show the beneficial effects of the disulfide isomerase DsbC and the prolyl isomerase FkpA on antibody folding and assembly [61].…”

“…Subsequently, the developed cell-free translation system was used for the in vitro display of antibody Fab fragments by using ribosome display [77]. In 2014, protein yields of cell-free synthesized trastuzumab full length antibody were further increased to gram per liter quantities by using a chaperoneenriched extract prepared from an engineered E. coli strain [61].…”

“…As nucleic acid templates may be translated at different rates-due to their codon usage and/or mRNA secondary structures-an empiric optimization of antibody heavy to light chain ratios may be necessary. Taking advantage of the open nature of cell-free reactions, the total amount as well as the ratio of antibody heavy to light chain encoding DNA templates can be varied systematically, in order to optimize the correct assembly of antibody polypeptide chains as successfully demonstrated by Yin et al and Groff et al [8,61].…”