Structural and biophysical studies of glycosylated proteins require recombinant protein samples of high quality and homogeneity. Production of glycoproteins relies mostly on eukaryotic protein expression systems 
. Protein-linked glycan chains are essential for protein folding and secretion, but they cause sample heterogeneity, which complicates protein crystallization and biophysical measurements, e.g. determination of molecular mass and oligomerization status 
. Inhibitors and mutations of N-acetylglucosaminyl-transferase I (GnTI) prevent the processing of N-linked glycans beyond the high-mannose type, leading to smaller and more homogeneous modifications 
. GnTI-negative HEK293 and CHO Lec 
cell lines have enabled the crystallization of a number of glycoproteins 
. High-mannose type glycans can be truncated efficiently to a single N-acetylglucosamine by endoglycosidase H, which usually does not affect protein stability, but often improves crystal growth 
Stable cell lines with good performance have integrated the recombinant transgene at a genetically stable hot spot of transcription. Preparative fluorescence-activated cell sorting (FACS) is very efficient for isolating such cell lines 
and was applied by us previously to glycosylation mutant CHO cells for crystallization of glycoproteins 
. However, preparative sorting of CHO cells growing in suspension can be challenging, especially if cell lines for several target proteins have to be established in parallel. Therefore, in this study, we combined cell lines carrying a fluorescent marker at a hot spot of transcription with targeted gene integration, thus allowing to derive production cell lines for arbitrary proteins from the same fluorescent master cell line in a single step ().
Genome engineering by recombinase-mediated cassette exchange (RMCE) allows targeted integration of transgenes precisely into defined expression hot spots of the host cell genome 
. RMCE with the recombinase Flp requires a master cell line ‘tagged’ at such a hot spot by a reporter gene cassette flanked by Flp recognition target (FRT) sites. The flanking FRT sites, the wild type and a synthetic variant, cannot recombine with each other. RMCE is achieved by co-transfecting the tagged master cell line with a targeting vector containing the gene of interest, flanked by the same pair of FRT sites, and a Flp expression vector (). A double-reciprocal crossover of the FRT sites leads to an exchange of the reporter with the gene of interest in the host cell genome. RMCE is thus practically irreversible, in contrast to recombination systems that use only a single recombination site 
. For antibiotic selection of recombinant cell lines, RMCE has been combined with a selection trap, consisting of a truncated antibiotic resistance marker that becomes complemented by the recombination event 
In the present study, GFP-positive master cell lines were established by preparative cell sorting that allows integrating genes of interest by RMCE with selection trap (). Stable production cell lines for 10 different proteins were established, including members of the heavily glycosylated lysosome-associated membrane protein (LAMP) family. The system was validated by expression, purification, deglycoslation and crystallization of different LAMP luminal domains.