Over-expression of secreted proteins from mammalian cell lines

Abstract

Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats.

Keywords: mammalian cell culture; protein expression; receptor; secreted protein.

Figure 1

Schematic representation of steps involved in over-expression of secreted proteins from stable mammalian cell lines. Expression constructs are initially evaluated by transient expression and SDS-PAGE in six-well plates. Approximately 48–72 h post transfection, cells are split directly into media containing selection reagent, for example. Cultures are maintained in selection media for approximately 8–14 days before visible colonies appear and can be cloned by traditional ring cloning techniques. Individual clones are expanded into six-well plates and tested for expression. Acceptable clones are expanded in larger dishes and an aliquot is preserved and cryogenically frozen. At this point, cell stocks are ready for large-scale growth.

Figure 2

SDS-PAGE analysis of various Fc-tagged proteins transiently expressed from 293 cells. One milliliter of media was harvested 48 h post-transfection and Fc-tagged fusion protein was immunoprecipitated with Protein-A Sepharose and electrophoresed on a 10% SDS-PAGE gel. Molecular weight markers are shown on the left.

Figure 3

Large-scale growth of adherent 293H cell lines. (A) A mini 37°C roller incubator can hold up to 40 expanded surface pleated roller bottles at a time (equivalent to 10 L of media). (B) For larger volumes, 293H cells can be efficiently grown in a fiber cell packed-bed bioreactor similar to the BioFlo 310 for weeks to months at a time using either fed-batch or perfusion culture techniques.