Gene Cloning

Production of Proteins from Cloned Genes 277
recombined it onto their chromosomes, they now become resistant to the
effect of MTX. The degree of resistance seen increases with the level of expression
of the DHFR gene, so this can be used to select for lines where the transforming
DNA has been integrated into a highly transcribed region in the
chromosome.
Further Reading
Many of the approaches described in this chapter for maximizing protein
expression, and overcoming the problems associated with trying to express
high levels of protein, have been commercialized. This means that the product
literature of the companies selling the particular solution is often an
excellent place to start to read. Examples of such literature are provided
below in addition to references to normal research papers. We’d better add
that their inclusion here does not imply endorsement of these particular
products!
The tac Promoter: A Functional Hybrid Derived from the trp and lac Promoters
(1983) De Boer HA, Comstock LJ and Vasser M, Proceedings of the National
Academy of Sciences of the United States of America, Volume 80 Pages 21–25.
An early and straightforward paper describing the construction of a hybrid strong
promoter for use in E. coli.
Tight regulation, modulation, and high-level expression by vectors containing the
arabinose pBAD promoter (1995) Guzman LM, Belin D, Carson MJ and Beckwith J,
Journal of Bacteriology, Volume 177 Pages 4121–4130.
The first description of the use of the tightly regulated pBAD promoter for protein
expression in E. coli.
http://www.emdbiosciences.com/docs/NDIS/inno01-000.pdf
A summary of the system which uses T7 RNA polymerase for tightly controlled expression
of proteins in E. coli.
Construction and characterization of a set of Escherichia coli strains deficient in
all known loci affecting the proteolytic stability of secreted recombinant proteins
(1994) Meerman HJ and Georgiou G, Biotechnology, Volume 12 Pages 1107–1110.
A description of the engineering and testing of multiply protease-deficient E. coli
strains, used here for the specific case of secreted recombinant proteins.
http://www.emdbiosciences.com/docs/NDIS/inno03-003.pdf
One particular system for helping to get soluble protein, by fusion to a soluble
partner.
http://www.invitrogen.com/downloads/PichiaBrochureP40.pdf and
http://www.invitrogen.com/content/sfs/brochures/710_021524_pyes_bro.pdf
Two brochures on yeast expression in S. cerevisiae and P. pastoris.
http://www.promega.com/vectors/mammalian_express_vectors.htm
Surf to this page for a look at maps and descriptions of the many different types of
mammalian expression system sold by one company.