The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.

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SSF3 is a CHO cell line adapted for growth in protein‐free medium. It grows in suspension unless serum‐derived attachment factors such as vitronectin are added to the medium. Serum‐independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor‐virus promoter. Substrate attachment and SSF3VN‐cell spreading could be prevented with an RGD peptide (arginine‐glycine‐aspartic acid) confirming that attachment is mediated by an intregrin receptor. Hormone‐inducible attachment could be blocked by glucocorticoid antagonist promegestone. All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum‐derived infectious agents. SSF3VN cells could be grown in protein‐free medium in solid‐phase large‐scale bioreactors. Application in microplates as used in high‐throughput screening was demonstrated in an assay of Ca2+ release from internal stores induced by agonist‐binding to recombinant human metabotropic glutamate receptor hmGluR1b. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65:523–528, 1999.