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P. TSIBOLI et al.
ceptible to proteolysis. It had been predicted by computer analysis that TthS14
has an extended structure. Protease inhibitors were therefore used at every step
of the purification procedure, which took place with the greatest possible speed
in the coldroom.
Additional attention must be paid to the conditions used during lysis of the
cells containing the overproduced protein to ensure that the protein is retrieved
in a soluble form. TthS14 is a very basic protein (pI = 11,94) and high ionic
strength conditions (0.8 M NaCl) were needed to avoid its precipitation after lysis
of the cells.
A problem peculiar to TthS14 was the presence of four cysteines. These might
form inter and/or intramolecular bonds leading to the aggregation of the protein.
During the purification procedure no aggregation took place probably because
the cysteines form the zinc finger domain with the zinc atom. Nevertheless,
(3-mercaptoethanol was added to protect the protein from oxidation.
By taking into consideration the problems common to overproduced proteins
and the individual characteristics of TthS14 it was possible to overproduce and
purify this small, highly basic protein with four cysteines.
4
Purification
The recombinant TthS14 protein was purified according to the following procedure: the BL21 E. coli cells carrying the TthS14 gene were harvested 3 h after
induction and suspended in the following buffer: 20 mM Tris-HCl pH 7.5, 10 mM
MgCl2, 50 mM NH4Cl, 0.8 M NaCl, 7 mM (3-mercaptoethanol and 1 mM PM SF.
After disruption of the cells by sonication, the cell lysate was subjected to two
successive centrifugations at 10000 x g for 20 mins and at 100000 x g for 3 h to
remove cell debris and ribosomes, respectively. The sodium chloride concentration in the lysis buffer was 0.8 M in order to maintain the overproduced protein
in soluble form. At lower concentrations of sodium chloride, namely 0.4 M and
0.6 M, the protein was detected not only in the supernatant of the 100000 x g centrifugation (S100) but also in the pellet (data not shown). Optimal recovery was
achieved at 0.8 M NaCl.
The final supernatant (S100) was dialysed against buffer containing 20 mM
Tris-HCl pH 7.5, 0.1 M NaCl, and applied onto a DEAE-Sepharose column equilibrated in the same buffer. The TthS14 protein was not retained by the column.
The flow-through fractions of the DEAE-Sepharose column were dialysed against
buffer containing 20 mM Tris-HCI pH 6.5, 0.1 M NaCl and applied on a CMSepharose column equilibrated in the same buffer. The TthS14 was eluted from
the CM-Sepharose at a concentration of 0.8 M NaCl by means of a NaCl concentration gradient (0.1-1.5 M) in the same buffer (Fig. 20.3). Finally, the fractions
that contained the S14 protein were concentrated by (NH4hS04 precipitation.
By following this procedure 15-20 mg of pure TthS14 were isolated from 45 g of
cells. At every purification step the protein was identified by Edman degradation
(after electrotransfer onto PVDF membranes (Choli et al., 1989» and by immunoblotting using the antiserum raised against the overproduced protein. The purified
protein was homogeneous as shown by SDS-PAGE analysis (Fig. 2004).
P. TSIBOLI et al.
ceptible to proteolysis. It had been predicted by computer analysis that TthS14
has an extended structure. Protease inhibitors were therefore used at every step
of the purification procedure, which took place with the greatest possible speed
in the coldroom.
Additional attention must be paid to the conditions used during lysis of the
cells containing the overproduced protein to ensure that the protein is retrieved
in a soluble form. TthS14 is a very basic protein (pI = 11,94) and high ionic
strength conditions (0.8 M NaCl) were needed to avoid its precipitation after lysis
of the cells.
A problem peculiar to TthS14 was the presence of four cysteines. These might
form inter and/or intramolecular bonds leading to the aggregation of the protein.
During the purification procedure no aggregation took place probably because
the cysteines form the zinc finger domain with the zinc atom. Nevertheless,
(3-mercaptoethanol was added to protect the protein from oxidation.
By taking into consideration the problems common to overproduced proteins
and the individual characteristics of TthS14 it was possible to overproduce and
purify this small, highly basic protein with four cysteines.
4
Purification
The recombinant TthS14 protein was purified according to the following procedure: the BL21 E. coli cells carrying the TthS14 gene were harvested 3 h after
induction and suspended in the following buffer: 20 mM Tris-HCl pH 7.5, 10 mM
MgCl2, 50 mM NH4Cl, 0.8 M NaCl, 7 mM (3-mercaptoethanol and 1 mM PM SF.
After disruption of the cells by sonication, the cell lysate was subjected to two
successive centrifugations at 10000 x g for 20 mins and at 100000 x g for 3 h to
remove cell debris and ribosomes, respectively. The sodium chloride concentration in the lysis buffer was 0.8 M in order to maintain the overproduced protein
in soluble form. At lower concentrations of sodium chloride, namely 0.4 M and
0.6 M, the protein was detected not only in the supernatant of the 100000 x g centrifugation (S100) but also in the pellet (data not shown). Optimal recovery was
achieved at 0.8 M NaCl.
The final supernatant (S100) was dialysed against buffer containing 20 mM
Tris-HCl pH 7.5, 0.1 M NaCl, and applied onto a DEAE-Sepharose column equilibrated in the same buffer. The TthS14 protein was not retained by the column.
The flow-through fractions of the DEAE-Sepharose column were dialysed against
buffer containing 20 mM Tris-HCI pH 6.5, 0.1 M NaCl and applied on a CMSepharose column equilibrated in the same buffer. The TthS14 was eluted from
the CM-Sepharose at a concentration of 0.8 M NaCl by means of a NaCl concentration gradient (0.1-1.5 M) in the same buffer (Fig. 20.3). Finally, the fractions
that contained the S14 protein were concentrated by (NH4hS04 precipitation.
By following this procedure 15-20 mg of pure TthS14 were isolated from 45 g of
cells. At every purification step the protein was identified by Edman degradation
(after electrotransfer onto PVDF membranes (Choli et al., 1989» and by immunoblotting using the antiserum raised against the overproduced protein. The purified
protein was homogeneous as shown by SDS-PAGE analysis (Fig. 2004).