274
2
Cloning
P. TSIBOLI et aI.
The TthS14 gene was amplified from Thermus thermophilus genomic DNA using
primers based on the known sequence of the TthS14 gene (Tsiboli and Choli
1995). The resulting 200 bp PCR product was cloned into the pETlld vector and
the recombinant plasmid (pETlld/TthSI4) was used for the transformation of
E. coli BL21(DE3)pLysS cells. Transformants were selected from Luria-Bertani
plates - supplemented with 40 IAg/ml ampicillin and 50 IAg/ml chloramphenicol -
analysed by restriction analysis and the positive clones were subjected to
sequencing. For more details see: Tsiboli et al. (1998).
3
Overproduction
During the overproduction of a protein several problems may occur. Firstly, the
protein might form inclusion bodies. From a purification standpoint, the accumulation of protein in an aggregated form can be advantageous. After lysing the
cells and centrifuging the resulting lysate the agreggated protein can be recovered
in the pellet fraction about 50 % pure, although mostly in an inactive form. The
protein in the inclusion bodies can be a mixture of monomeric and multimeric
forms, both reduced and oxidized. The major problem then becomes one of
recovering biologically active protein in high yield. In order to accomplish this,
the protein in the inclusion bodies must be solubilized, refolded and purified in
a specific order. The common stages in processes designed to recover biologically
active, soluble protein from such aggregates include
(1) cell lysis,
(2) isolation of inclusion bodies,
(3) solubilization of protein in inclusion bodies and
(4) refolding of solubilized protein.
Sometimes the overproduction of a protein at lower temperatures alleviates the
problem and for this reason it is recommended to grow the cells at different temperatures, to control - after centrifugation of the lysate - the supernatant by SDS
electrophoresis and finally to chose the right temperature for cell growth.
After all these controls we did not detect the S14 protein in the centrifugation
pellet and therefore decided to grow the transformed E. coli cells at 37 DC, which
is the optimal temperature for E. coli.
For vectors carrying the lac-z promoter, namely for IPTG inducible vectors,
two factors that have to be taken into account are the IPTG concentration and the
absorption of the culture where the induction takes place. In Fig. 20.1 we show
that at concentrations of IPTG over 1.0 mM there were no significant differences
in the amount of the overproduced cloned protein. The chosen absorption for the
induction was between 0.7-0.9 because at lower values the protein was in very
low yield (Fig. 20.2).
In the case of small proteins, such as TthS14, proteases might partially or fully
digest the overproduced protein. Extended protein structures are even more suc-
2
Cloning
P. TSIBOLI et aI.
The TthS14 gene was amplified from Thermus thermophilus genomic DNA using
primers based on the known sequence of the TthS14 gene (Tsiboli and Choli
1995). The resulting 200 bp PCR product was cloned into the pETlld vector and
the recombinant plasmid (pETlld/TthSI4) was used for the transformation of
E. coli BL21(DE3)pLysS cells. Transformants were selected from Luria-Bertani
plates - supplemented with 40 IAg/ml ampicillin and 50 IAg/ml chloramphenicol -
analysed by restriction analysis and the positive clones were subjected to
sequencing. For more details see: Tsiboli et al. (1998).
3
Overproduction
During the overproduction of a protein several problems may occur. Firstly, the
protein might form inclusion bodies. From a purification standpoint, the accumulation of protein in an aggregated form can be advantageous. After lysing the
cells and centrifuging the resulting lysate the agreggated protein can be recovered
in the pellet fraction about 50 % pure, although mostly in an inactive form. The
protein in the inclusion bodies can be a mixture of monomeric and multimeric
forms, both reduced and oxidized. The major problem then becomes one of
recovering biologically active protein in high yield. In order to accomplish this,
the protein in the inclusion bodies must be solubilized, refolded and purified in
a specific order. The common stages in processes designed to recover biologically
active, soluble protein from such aggregates include
(1) cell lysis,
(2) isolation of inclusion bodies,
(3) solubilization of protein in inclusion bodies and
(4) refolding of solubilized protein.
Sometimes the overproduction of a protein at lower temperatures alleviates the
problem and for this reason it is recommended to grow the cells at different temperatures, to control - after centrifugation of the lysate - the supernatant by SDS
electrophoresis and finally to chose the right temperature for cell growth.
After all these controls we did not detect the S14 protein in the centrifugation
pellet and therefore decided to grow the transformed E. coli cells at 37 DC, which
is the optimal temperature for E. coli.
For vectors carrying the lac-z promoter, namely for IPTG inducible vectors,
two factors that have to be taken into account are the IPTG concentration and the
absorption of the culture where the induction takes place. In Fig. 20.1 we show
that at concentrations of IPTG over 1.0 mM there were no significant differences
in the amount of the overproduced cloned protein. The chosen absorption for the
induction was between 0.7-0.9 because at lower values the protein was in very
low yield (Fig. 20.2).
In the case of small proteins, such as TthS14, proteases might partially or fully
digest the overproduced protein. Extended protein structures are even more suc-