Sustainability and Stability - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 823

TABLE 16.3 Strong Escherichia coli Promoters

Promoter Induction Method Characteristics'

lacUV5 Addition of isopropyl- b - D -

thiogalactoside

(About 5%)

tac As above Induction results in cell death ( > 30%)

Ipp-OmpA As above Suitable for secreted proteins (20%)

Ipp P -5 As above Strongest E. coli promoter (47%)

trp Tryptophan starvation Relatively weak (around 10%)

Growth at 42 C

l p L See text ( > 30%)

l p L /cl up Addition of tryptophan Easily inducible in large-scale

production (24%)

10-Min incubation at 42 C

att-nutL-p-att-N No product is synthesized before

induction (on/off promoter)

T7 promoter Addition of isopropyl- b - D -

thiogalactoside or viral infection

As above ( > 35%), low basal levels

T4 promoter Viral infection Method of induction inhibits product

degradation

phoA Phosphate starvation Induction in large-scale production is

complicated

Typical values of accumulated product as a percent of the total protein of induced cells are given in parentheses.
Source: G. Georgiou, AIChE J. 34:1233 (1988).

the production of a fusion protein can be made. Typically, a small part of a protein native to
the host cell is fused to the sequence for the target protein with a linkage that can be easily
cleaved during downstream processing. Also, fusion proteins may be constructed to facilitate
downstream recovery by providing a “handle” or “tail” that adheres easily to a particular
chromatographic medium.
Another approach to preventing intracellular proteolysis is to develop a secretion vector in
which a signal sequence is coupled to the target protein. If the protein is secreted in one host,
it will usually be excreted in another, at least if the right signal sequence is used. Replacement
of the protein's natural signal sequence (e.g. from a eukaryotic protein) with a host-specific
signal sequence can often improve secretion. The secretion process is complicated, and the
fusion of a signal sequence with a normally nonsecreted protein (e.g. cytoplasmic) does
not ensure secretion, although several cases of secretion of normally cytoplasmic proteins
have been reported. Apparently, the mature form of the protein contains the “information”
necessary in the secretion process, but no general rules are available to specify when coupling
a signal sequence to a normally cytoplasmic protein will lead to secretion.
To ensure the genetic stability of any construct and to aid in the selection of the desired
host e vector combination, the vector should be developed to survive under selective growth
conditions. The most common strategy is to include genes for antibiotic resistance. The
common cloning plasmid, pBR322, contains both ampicillin and tetracycline resistance.