5.5 Protein Expression and Purification
5.5.3 Expression and Purification of CbiX^H
5.5.3.1 Solubility o f CbiX^H
In order to assess the solubility of CbiX^H the soluble and insoluble fractions of an overnight culture of JMlOl/pRBXA were prepared by sonication and centrifugation.
The strain was grown for 12 hours in 4 x 750ml LB+A, with protein expression being induced w ith 0.2m M IPTG for a further 2 hours. The cells w ere harvested by centrifugation at 12,500 x g for 15 minutes at 4°C and the pellet resuspended in 20ml of 50mM Tris-HCl (pH7.5) containing ImM EDTA, 5mM B-mercaptoethanol and 10% (v/v) glycerol. This suspension was sonicated and the soluble extract was separated by
centrifugation at 40,000 x g for 30 minutes at 4°C. Successive ammonium sulphate 'cuts'
o f 0-20%, 20-40% and 40-60% were taken from the soluble fraction of JM lO l/pRBX A
and all cell extracts were assessed by SDS-PAGE (see Figure 5.12).
The CbiX'^H protein is expressed in JMlOl/pRBXA such that it makes up approximately
25% of the total cell protein (Fig. 5.12, lane 1). In contrast to the native CbiX protein and
the CbiX^H variant however, a substantial proportion of C biX "^ is present in the insoluble extract of the JM lOl/pRBXA (lane 2). Very little (if any) of this 27kDa protein is soluble (lane 3), and no significant purification of any remaining CbiX^^H was achieved through ammonium sulphate precipitation (lanes 4-9).
CbiX^H was insoluble when bacteria expressing the protein were incubated at 37°C. This phenom enon also occurred in smaller-scale experiments when the same bacteria were incubated at 30°C. The presence of 10% glycerol in the buffer used prior to sonication had no effect on the solubility of CbiX^H The implications of these findings will be discussed further in the Summary (5.6).
66 45 36 29 24 20 1 2 3 4 M 5 6 7 8 9 10
Figure 5.12 Assessment of the Solubility of CbiX^H
The JM 101/pRBXA strain was incubated overnight in 3 litres of LB+A, and IPTG was added (final concentration lOOpM) at 2h before harvest. Bacteria were harvested by
centrifugation at 12,500 x g, and re-suspended in 20ml of 50mM Tris-HCl (pH7.5)
containing ImM ED TA , 5mM B-m ercaptoethanol and 10% (v/v) glycerol. The suspension was sonicated for 4 x 1 m inute bursts on ice and the sonicate was centrifuged to separate soluble from insoluble material. The pellet was re-suspended in 10ml buffer (see above), w hilst the soluble extract was subjected to differential precipitation with ammonium sulphate (’cuts').
Key to cell fractions:
1 Total extract pre-sonication
2 Total cell protein
3 Insoluble cell protein
4 Soluble cell protein
5 pellet from 0-20% cut of soluble material
6 soluble extract from 0-20% cut
7 pellet from 20-40% cut
8 soluble extract from 20-40% cut
9 pellet from 40-60% cut
10 soluble extract from 40-60% cut
5.6
Summary
In vivo, it has been shown that native CbiX is able to transform precorrin-2 into sirohaem and must have inherent dehydrogenase and ferrochelatase functions. The poly(histidine) m otif o f CbiX has been implicated in the ferrochelatase activity o f the protein and is certainly likely to bind endogeous metal ions. In fact, the purification of CbiX by nickel- binding chromatography demonstrates this ability.
In order to investigate further the role of the poly(histidine) m otif in CbiX, two mutant variants were generated, where the m otif was either removed or truncated. The former
mutant was made by removing a Pstl fragment from cbiX which resulted in the deletion of
59 amino acids from the C-terminus of CbiX, including the histidine tail. The latter variant was generated as an expression cassette by PCR which was then cloned into pKK223.3.
To test the ability of the mutant variants of cbiX to function 'in vivo', they were cloned in
tandem with a uro'gen III methylase from P. denitrificans (cobA^^) in pKK223.3. The
plasmids were investigated for their ability to complement E. coli 302Aa. It was found that
whereas CbiX^H retained the function ascribed to CbiX, the protein missing the C-terminal 59 amino acids, C b iX ^ was not functional.
The two mutants of CbiX have been over-expressed in E. coli JM lO l. The proteins were
over-expressed to approximately 10% total protein and migrated, upon electrophoresis, at the expected rate. One of the mutants, CbiX^^, has been purified to homogeneity using a method sim ilar to that used for purification of CbiX. Thus CbiX^H was purified by H is'B ind nickel-binding chromatography, followed by anion exchange chromatography. W hilst CbiX was eluted with a sodium chloride concentration of approximately 300mM, CbiX6H is eluted in 550mM NaCl.
The CbiX^H protein was insoluble in E. coli JM lO l. This raises important questions with
respect to the com plem entation results obtained with the c b iX ^ ^ gene - the lack of
complementation of E. coli 302Aa seen with pER192 {^YJA.cobA^^.cbiX^) may be due to
the low solubility of CbiX^H vivo. However, this in turn may be due to the lack of the
poly(histidine) motif.
Just as the shortened poly(his) sequence of CbiX^H may be responsible for the reduced amount of proteolysis seen in early stages of purification (by perhaps causing the protein to adopt a tighter conformation more resistant to proteolysis), the complete absence of the m otif may expose hydrophobic residues below the surface of the CbiX protein.