Streamlining Homogeneous Glycoprotein Production for Biophysical and Structural Applications by Targeted Cell Line Development

Abstract

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP ‘master’ cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).

Generation of monoclonal antibody-producing mammalian cell lines

“…Cell line development using targeted integration has been demonstrated in a few studies based on sitespecific recombinases, such as Flp, Cre and PhiC31 [152,[154][155][156][157][158][159][160][161][162]. Identification of a hotspot in the genome by classical transfection of a screening vector, and subsequent screening of single copy integration, high expression level, and stability is first carried out.…”

Better and faster: improvements and optimization for mammalian recombinant protein production

“…These include the human retina-derived Per.C6 and amniocyte-derived Cap-T lines, which are capable of very high-density growth (~5–15*10 7 cells/ml) that supports concomitant increases in protein yield and decreased media costs (reviewed in [3, 4]). Cell types may also be chosen, or engineered, to alter the extent of post-translational modifications such as glycosylation, lipidation, sulphation etc., which can modulate the activity of the target protein (e.g., 10–20 fold in the case of an anti-CD20 antibody) [5, 6]. Unfortunately these post-translational modifications, while essential for function, may be inversely correlated with the success of structural studies, as increased heterogeneity can adversely affect crystal packing [7]; but numerous options are available for the mitigation of these challenges.…”

3.4 Rational Approaches for Transgene Expression: Targeted Integration and Episomal Maintenance

“…Schucht and coworkers [84] used high expressing master cell lines to express G protein-coupled receptors (GPCRs). Wilke et al used RMCE in mutant CHO Lec3.2.8.1 cells to produce glycoproteins with the well-established glycosylation pattern in a homogenous form [93].…”