Consideration in plasmid design to avoid process problems

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12 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Consideration in plasmid design to avoid process problems

R.S. Sengar, Reshu Chaudhary and Kalpana Sengar

Tissue culture laboratory, College of biotechnology
, Sardar Valla Bhai Patel University of Agriculture &
Technology, Meerut (U.P.)


When we design vectors for genetic engineering , we are concerned with elements that control plasmid
copy number, the level of target

gene expression, and the nature of the gene product, and we must allow
for the application of selective pressure (e.g.,
antibiotic resistence).The vector must also be designed to be
compatible with the host cell.Different origins of replication exist for various plasmid.The origin often
contains transcript that regulate copy number.Different mutation in these regulatory
transcripts will yield
greatly different copy number.In some cases, these transcripts have temperature sensitive mutation, and
temperature shift can lead to runaway replication in which plasmid copy number increases until cell death
occurs
.

Total protein p
roduction depends on the both the numbers of gene copies (e.g. the number of plasmid) and
the strength of the promoter used to control transcript from these promoters.Increasing copy number while
maintaining a fixed promoter strength increases protein pro
duction in a saturable manner.Typically ,
doubling copy numbers from 25 to 50 will increase protein production twofold, but an increase from 50 to
100 will increase protein production less than twofold .If the number of replicating units is above 50, pure

segregational plasmid loss is fairly minimal .Most useful cloning vector in E. coli have stable copy number
from 25 to 250.

Many promoters exist .Some of the important ones for use in e.coli are listed in table.
An ideal promoter
would be both very stro
ng and tightly regulated.A zero basal level of protein production is desirable ,
particularly if the target protein is toxic to the host cell.A rapid response to induction is desirable, and the
inducer should be cheap and safe.Although temperature inducti
on is often used on a small scale, thermal
lags in a large fermentation vessel can be problematic . Increased temperature may also activate a heat

shock response and increased levels of proteolytic enzymes.Many chemical inducers are expensive or might
ca
use health concern if not removed from the product .Some promoters respond to starvation for a nutrient
(e.g. phosphate, oxygen, and energy), but the control of induction with such promoters can be difficult to
do precisely.The recent isolation of a promo
ter induced by oxygen depletion may prove useful, because
oxygen levels can be controlled relatively easily in fermenters.


Promoter

Induction method

characteristics

Lac UV5

Addition of IPTG

(about 5%)

tac

As above

Induction results in cell death (

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Strongest E.coli promoter (47%)

trp

Tryptophan starvation

Relatively weak (around 10 %)

λ
p
L

Growth at 42

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(

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trp

Addition of tryptophan

Easily inducible in large
-
scale

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(24%)

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-
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constructed to facilit
ate 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 tar
get protein. If the protein is secreated 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 an eukaryotic protein) with a host

specific sign
al sequence can often improve secretion.

The secretion process is complicated, and the fusion of signal sequence with a normally nonsecreted
protein(e.g. cytoplasmic) does not ensure secretion, although several cases of secretion of normally
cytoplasmic

protein have been reprted .Apparently, the mature form of the pritein contain 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

secreation.

To ensure the genetic stability of any construct and to aid in the selection of the desired host

vector
combination, the vector should be developed to survive under selective growth conditions. The most
common strategy is to include gene for
antibiotic resistance .The common cloning plasmid, pBR322,
contains both ampicillin and tetracycline resistance. Multiple resistence gene are an aid in selecting for
human


designed modifications of the plasmid.

Another strategy for selection is to place on the vector the genes necessary to make an essential metabolite
(e.g. an amino acid). If the vector is placed in a host that is auxotrophic for that amino acid , then the vector
complement the host.In a medium
without that amino acid , only plasmid

containing cells should be able
to grow.B
ecause the genes for the synthesis of the auxotrophic factors can be integrated into
thechromosome or because of reversion on the parental chromosome, double auxotrophs are of
ten used to
reduce the probability of nonplasmid
-

containing cells outgrowing the desired construction.

One weakness in both of these strategies is that , even when the cell loses the plasmid , the plasmid

free
cell will retain for several division enoug
h gene product to provide antibiotic resistence or the production of
an auxotrophic factor.Thus , cell that will not form viable colonies on selective plates (about 25 generations
are required to form a colony) can still be present and dividing in a large


scale system.These plasmid free
cells consume resources without making product.

Another related problem . particularly in large
-
scale system, is that plasmid containing cells may protect
plasmid

free cells from the selective agent .For example, auxotrop
hic cells with a plasmid may leak
sufficient levels of the auxotrophic factor that plasmid

free cells can grow.With and antibiotic, the enzyme
responsible for antibiotic degradation may leak into the medium.Also the enzyme may be so effective, even
when r
etained intracellularly, that all the antibiotic is destroyed quicly in a high
-

density culture, reducing
the extracellular concentration to zero.Although genes allowing the placement of selective pressure on a
culture are essential in vector development ,

trhe engineer should be aware of limitation of selective
pressure in commercial

scale systems.

The other useful addition to plasmid construction is the addition of element that improve plasmid
segregation.Examples are the so
-

called par and cer loci.Thes
e elements act positively to ensure more even
distribution of plasmids.The mechanisms behind these elements are incompletely understood, although
they may involve promoting plas
mid

membrane complex (the par locus) or decreasing the net level of
multimeri
zation (the
cer

locus).multimerization decreses the number of independent , onheritable units,
thus increasing the probability of forming a plasmid free cell.

Any choice of vector construction must consider host cell characterstics.Proteolytic degradation
may not be
critical if the host cell has been mutated to inactivate all known proteases.Multimerization can be reduced
by choosing a host with a defective recombination system.However, host cells with a defective
recombination system tend to grow poorly.Ma
ny other possible host cell modification enter into
consideration of how to best construct a vector for a commercial operation.

These qualitative ideas allow us to anticipate to some extent what problem may arise in the maintenance of
genetic stability an
d net prtein expresiion.However, a good deal of research has been done on predicting
genetic instability.