Biomedical Engineering Reference
In-Depth Information
Extracellular release of target proteins is most preferable. Normally, E. coli does not excrete
protein (colicin and hemolysin are the two exceptions), but a variety of schemes to obtain
excretion in E. coli are being developed. Strategies usually involve either trying to disrupt
the structure of the outer membrane or attempting to use the colicin or hemolysin excretion
systems by constructing a fusion of the target protein with components of these excretion
systems. Excretion without cell lysis can simplify recovery and purification even more
than secretion alone, while achieving the same advantages as secretion with respect to
protein-processing. Excretion also facilitates the potential use of continuous immobilized
cell systems.
Excretion of normally cytoplasmic or human-designed proteins is problematic. There are
reports for the excretion of normally cytoplasmic proteins, but the general principles for the
extension of excretion to cytoplasmic proteins are still being developed. The excretion of nor-
mally secreted proteins can be obtained in E. coli (and other cells) even when the protein is
derived from animal cells. However, extension to nonsecreted proteins is difficult.
The lack of established excretion systems in E. coli has led to interest in alternative expression
systems. Also, in some cases, patent considerations may require the use of alternative hosts.
14.7.2. Gram-Positive Bacteria
The gram-positive bacterium, Bacillus subtilis , is the best studied bacterial alternative to
E. coli . Since it is gram positive, it has no outer membrane, and it is a very effective excreter
of proteins. Many of these proteins, amylases and proteases, are produced commercially
using B. subtilis . If heterologous proteins could be excreted as efficiently from B. subtilis ,
then B. subtilis would be a very attractive production system. However, B. subtilis has
a number of problems that have hindered its commercial adoption. A primary concern has
been that B. subtilis produces a large amount and variety of proteases. These proteases can
degrade the product very rapidly. Mutants with greatly reduced protease activity have
become available, but even these mutants may have sufficient amounts of minor proteases
to be troublesome. Bacillus subtilis is also much more difficult to manipulate genetically
than E. coli because of a limited range of vectors and promoters.
Also, the genetic instability of plasmids (Chapter 15) is more of a problem in B. subtilis than
in E. coli . Finally, the high levels of excretion that have been observed with native B. subtilis
proteins have not yet been obtainable with heterologous protein (i.e. foreign proteins
produced from recombinant DNA).
Other gram-positive bacteria that have been considered as hosts include Streptococcus cre-
moris and Streptomyces sp. These systems are less well characterized than B. subtilis .
Both gram-negative and gram-positive bacteria have limitations on protein-processing
that can be circumvented with eukaryotic cells.
14.7.3. Lower Eukaryotic Cells
The yeast, S. cerevisiae , has been used extensively in food and industrial fermentations and
is among the first organisms harnessed by humans. It can grow to high cell densities and at
a reasonable rate (about 25% of the maximum growth rate of E. coli in similar medium). Yeast
is larger than most bacteria and can be recovered more easily from a fermentation broth.
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