Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence‐activated cell sorting

Abstract

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

A streamlined implementation of the glutamine synthetase-based protein expression system

“…It needs to be borne in mind, however, that if needed, better expressing lines could be obtained by FACS-selecting the top 10% of expressers. It is possible that the synthesis of an additional protein (eGFP) may burden the cell machinery in the case of the FcERα-IRES-eGFP-GS-pOPINEE12G transfected clones and lines, accounting for the reduced expression [ 13 ].…”

“…No significant advantages were obtained by using dual promoters to express the two proteins, or 2A sequences. The success of the new approach likely hinges on the inherent strength of the expression system since previous attempts to utilize fluorescence-based selection used two [ 12 ] or more (up to 5) [ 13 ] rounds of FACS selection. The ease with which close-to-optimal expression is obtainable in a single step with the new approach, coupled with the target protein-independent selection of expressing cells, offsets some of the risk in producing hard-to-assay proteins, as exemplified by our expression of biotinylatable CCL18.…”

“…green fluorescent protein (GFP), as second selectable markers [ 11 ]. Previously, implementation of this approach has involved either two [ 12 ] or more (up to five) [ 13 ] rounds of FACS selection of the GFP-expressing cells, resulting in these methods still being labor-intensive and taking six months or longer. This effort is justified in the context of the industrial expression of therapeutic proteins, where production can be scaled and repeated indefinitely.…”

SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level

“…Other methods rely on the coexpression of a fluorescent protein (e.g., GFP) with the gene of interest, either through an internal ribosome entry site (IRES), using polycistronic vectors or by co-transfection. [6][7][8][9][10][11][12][13][14] However, limitations in the secretory pathway, protein folding or different efficiencies in translation may affect the correlation of expression of the reporter protein and the gene of interest. These limitations are partially overcome by using a transmembrane reporter protein co-expressed with the protein of interest, such as using an IRES in the expression cassette, [14][15][16][17][18] or due to a non-optimal start codon, 19 even if an expression bias, which could be caused by uncorrelated expression of reporter and gene of interest due to different folding kinetics, may still be possible.…”

Generation of Flp-in tm -ready DG44 and Lec 3.2.8.1 CHO Cell Lines for Quick And Easy Constitutive Protein Expression

“…Thus these cells lack the ability to attach complex N-type glycans to proteins, but still retain the less complex O-glycosylation pathways; the end result is that cells homogeneously glycosylate proteins. This property is highly desired by protein crystallographers, since the homogeneously glycosylated proteins facilitate crystallization [8,9]. Another important selectable cell cloning line, CHO-DG44, was developed by Chasin and Urlaub [10].…”