Methods and means relating to quiescent cells and uses thereof

Details Methods and means relating to quiescent cells and uses thereof Methods and means relating to quiescent cells and uses thereof

1999-01-22

2001-02-20

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S476000, C435S252300, C435S252330

Reexamination Certificate

active

06190867

ABSTRACT:

The present invention relates to cells in culture. In particular, it relates to bacterial cells in broth culture which may produce a heterologous (cloned) gene product, and synchronisation of cell division. Methods and means are provided for inducing and capitalising on quiescence of cells.
The development of recombinant DNA technology over the last 20 years has made it possible to identify and isolate genes from any organism and express products in bacteria (the most common host organism is the enteric bacterium
Escherichia coli
). To achieve this, the gene is first inserted into a vector and then introduced into the bacterium by a method such as transformation or electroporation. Cloning and expression vectors are generally derivatives of plasmids: autonomously-replicating DNA circles which are found extensively in natural populations of bacteria. Typically, vectors carry some kind of marker to facilitate selection of vector-containing cells (for example an antibiotic resistance gene) and expression signals which direct the host bacterium to synthesise exogenous genes.
A number of factors may reduce the efficiency with which the products of cloned genes are expressed in a bacterial host:
Many plasmid cloning vectors have copy numbers far in excess of that of their naturally occurring progenitors. For example, natural plasmid ColE1 has a copy number of 20-30 compared with 100-200 for vector pUC18. This increase in DNA loading on the cell gives a selective advantage to any cell which loses the plasmid and means that plasmid-free cells accumulate rapidly in the culture.
If the product gene is expressed constitutively (i.e. throughout the growth of the culture) or if it is expressed from an inducible promoter which cannot be turned off completely during the early stages of culture growth (which is a common situation) there will be even more metabolic demands placed upon plasmid-bearing cells. This will give an advantage to plasmid-free cells or to cells containing rearranged vectors which no longer express the product gene. If these arise early in the culture, the consequences for productivity will be serious. One solution to these problems is to arrange for vector copy number to be low initially and to increase when expression of the product gene is required. It is, for example, possible to increase copy number by down-regulating synthesis of the replication inhibitor or by up-regulating synthesis of an initiator protein (Rep) or replication primer.
Present technology requires that cloned genes are expressed in actively-growing cells. The cellular machinery required for transcription and translation of the recombinant gene is required also for the expression of genes essential for the growth of the host cell. Furthermore, the economy of the cell is devoted largely to the creation of biomass. There is thus a conflict between the requirements of the biotechnologist and the bacterium.
The metabolic stress imposed by the expression of a recombinant gene invariably reduces the growth rate and viability of the host cell. The higher the copy number of the cloning vector and the expression level of the cloned gene, the greater the effect. Cells which have lost the cloning vector or have deleted or rearranged the cloned gene will almost invariably out-grow the original cell-type, reducing yield and purity of the product.
In addition to problems arising from the metabolic load placed upon the cells by the need to replicate the plasmid and to transcribe and translate its genes, the cloned gene product may be toxic to the host cell or interfere with its growth and division. An illustration of this phenomenon is provided by the
E. coli
LacY permease which is responsible for transport of lactose across the cell membrane. Over-expression of LacY is lethal to the cell. Similar problems are likely to be experienced if membrane proteins from any source are expressed-in a bacterial host. Problems might also arise if the product of the cloned gene interferes with replication of the bacterial chromosome, transcription of genes essential to the growth and division of the host cell, or disrupts other vital processes. Many adverse effects on growth of the bacterial host may be avoided if the protein is expressed in non-growing (quiescent) cells.
Expression of the cloned gene is simultaneous with the expression of thousands of genes located on the host chromosome. The recombinant product is therefore likely to represent a relatively small part of total cell protein, especially if the copy number of the cloning vector is not very high or if the product of the cloned gene is harmful to the host bacterium.
An aspect of the present invention utilises viable but quiescent (non-growing) cells in which the expression of chromosomal genes is repressed but expression of vector- (e.g. plasmid-) borne genes is allowed.
To our knowledge there has been no previous serious attempt to use quiescent cells as factories for the production of recombinant gene products. Pre-existing methods to inhibit cell growth almost invariably have undesirable side effects such as induction of the SOS response (which increases the mutation rate and causes cell filamentation) or interference with aspects of host metabolism which are required for expression of the cloned gene.
The present invention in various embodiments makes use of the rcd gene transcript of
E. coli
plasmid ColE1 or the equivalent from another bacterium or plasmid. The rcd transcript is produced in nature by expression from a promoter (P
cer
) within the cer site of dimerised ColE1 plasmids (Patient & Summers, 1993; FIG.
1
). It is one of the ways by which ColE1 achieves a high degree of stability in a population of cells.
The formation of plasmid multimers (through inter-molecular recombination) is a major cause of instability of high copy number plasmids and cloning vectors (Summers, 1991) and therefore a further problem for the biotechnologist. Multimerisation reduces the plasmid copy number and, because the plasmids are distributed randomly between the daughter cells, it increases the frequency at which plasmid-free cells arise. A dividing cell containing 40 plasmid monomers has a probability of 10
−12
of producing a plasmid-free daughter. In a cell which contains 20 dimers the probability is increased one million fold to 10
−6
Plasmid-free cells typically grow faster than those carrying plasmids and, as a result, the production of a few plasmid-free cells can be followed rapidly by the virtual disappearance of plasmid-containing cells. Such “segregational instability” can be a serious problem in large-scale culture where it is impractical to maintain a plasmid by selective pressure (e.g. by the addition of antibiotics to the culture medium).
Some natural plasmids are extremely stable compared with the majority of man-made cloning vectors. ColE1 (the plasmid upon which many cloning vectors are based) achieves its stability in a population of cells by action of three systems:
(a) Colicin Production. ColE1 carries a gene for the toxic colicin E1 protein (which is synthesised by bacteria and released into the growth medium) and a second gene which confers immunity to colicin. Cells which lose the plasmid are susceptible to killing by exogenous colicin because they cannot produce the “antidote” to the toxin. Cells which retain the plasmid are immune to the killing effect of the toxin.
(b) Conversion of Multimers to Monomers. Multimers are resolved to monomers by site-specific recombination. This process requires a 250 bp section of DNA in the plasmid (the cer site; Summers & Sherratt, 1984) and at least four proteins encoded by the host bacterium (XerC, XerD, ArgR and PepA). Recombination is unidirectional i.e. it converts dimers to monomers but not vice versa.
(c) Growth Inhibition of Multimer Containing Cells. It has been shown that plasmid dimers replicate at twice the rate of monomers because they contain two replication origins. As a direct consequence, if a single plasmid dimer is formed by homologous recombination, dimer-only cells will app

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