bioreactor studies of heterologous protein production by ...

2.2.1. Genetic Factors
2.2.1.1. Pidd DNA Sequenees
Plasmid stability may be enhanced by including stabilising gens in plasmid vectors. The 2
prn DNA-based vectors are the best-amiysed vector sysams in S. cerevisiae. Two
replication loci (REPI and REP2) of the 2 pm are responsibonsibIe for stability of the native
plasmid during replication (Panchal, 1987). Futcher and Cox (1984) have made use of this
to constmct 2 pm-based pst plasmid vecton. 'Ihey showed that the plasmids coniaining
both REPI and REP2 are considerably more stable than those lacking one or both loci.
Inclusion of chromosornai centmmere ngions in vectors was also used to construct stable
plasmids. One such plasinid YRp7, containhg autonomous replication sequenees (ARSs),
is fairly unstable in S. cerevisiae; however, once a centmmue region, CEN. carrying
panition fwction is inserted into YRp7. the plasmid becornes stable (Clarke and Carbon,
1980). Less than 5% of such vectors are lost &r 20-40 generations under nonselective
conditions. Plasmids can be stabWd by jouiing with a particular DNA segment that
pmvides the good partition function for E. coli (Skogrnan et al., 1983). When the
partition locus of piasmid pSClOl was added to both pBR322 and pACYC184 plasmids,
the stabilities of the resulting plasmid increased by at 3- to 10-fold. For the yeast system,
the STB (REP3) sequefice. in conjunction with the REPI and REP2 gene produas
encoded by the endogenous 2 pm plasmid? ensures a high degree of pdtion stability
(Reynolds et ai., 1987). in another mdy, the insenion of a k cos-contalliing DNA
sequence into one of the pSa plasmids incnased the fimess and the growth rate of the host
tek carrying this h. cos pSa conmct (Edlin et al.. 1984). While Ogura et aï. (1983)