Page 30 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 19

coprecipitated proteins, followed by treatment with difference in the redox state between E. coli and
detergents and denaturants such as urea, and finally eukaryotic cells may also affect protein solubility.
extensive dialysis in a suitable buffer containing This has been demonstrated by the fact that GST
refolding additives (salts, chaotropes, redox agents). fusions produced in E. coli bind to the glutathione
For proteins containing disulphide bonds, redox Sepharose beads with greater efficiency than similar
systems need to be included in the renaturation GSTfusionsproducedinmammaliancells. Thesame
buffer (Creighton, 1986). Although proved to be authors demonstrated that coproduction of bacterial
successful for relatively small proteins and polypep- thioredoxin (Trx) is more effective than the GroE sys-
tides, it cannot be guaranteed that larger proteins tem in producing soluble protein. Coexpression of
will refold into their native conformations. Kits one or more of the three different types of foldase
for optimizing the refolding conditions of inclusion (disulphide oxidoreductase (DsbA) and disulphide
body products are available from Takara Mirus Bio isomerase (DsbC); peptidyl prolyl isomerases (PPIs)
Madison Wisconsin (www.takaramirusbio.com). and protein disulphide isomerase (PDI)) could lead
The iFOLD™ is a new system for determin- to higher levels of soluble protein.
ing the optimum conditions for recombinant
protein refolding marketed by Novagen Merck 1.3.3.2 Refolding chromatography with
Biosciences (www.novagen.com). The System pro- minichaperones
vides a comprehensive set of conditions for tar- Peptides consisting of residues from GroEL immo-
get protein refolding based on the REFOLD bilized on agarose have proved effective minichap-
database (http://refold.med.monash.edu.au) in a erones (Altamirano et al., 1997). The procedure used
96-well plate format amenable for high-throughput both column chromatography and batch-wise meth-
automation. ods to renature an insoluble protein from an inclu-
sion body, refold apparently irreversibly denatured
1.3.3.1 Coproduction of the target protein with proteins, and to recondition enzymes that have lost
chaperones or foldases activity on storage. Fragments were immobilized
The formation of inclusions may be avoided in the by two methods: Ni-NTA resin and CNBr-activated
firstplacebyengineeringthesecretionoftheproduct Sepharose 4B.
into the periplasm. Protein folding in vivo is facili-
tated by a group of molecular chaperones belonging 1.3.4 Purification
to conserved families of proteins. These include the
Hsp100 (ClpA, ClpB, ClpP), Hsp90 (HtpG), Hsp70 The popular use of fusions and/or generic tags
(DnaK), Hsp60 (GroEL), and α-crystalline-like small obviates the need for extensive, time-consuming,
heat-shock proteins (IbpA, IbpB). Chaperones inter- multistep purification except where their use is
act transiently with non-native protein substrate, undesirable due to the loss of structural informa-
GroEL and DnaK, together with their cochaperones tion through tag interference, loss of solubility after
(GroES for GroEL; DnaJ and GrpE for DnaK), main- cleavage, or simply prohibitive cost of proteases. In
tain denatured proteins in non-aggregated states an optimally-designed purification scheme it should
whilst assisting their refolding by a mechanism of be possible to achieve a high-level of purity in fewer
recurrent ATPase-driven cycles of substrate binding than four key stages without compromising percent-
and release. age yield. To do this, the physicochemical properties
If strong promoters such as T7 are used and of the target protein should be well defined and
the level of functional GroESL does not increase a rapid, reliable assay developed to monitor the
proportionally, correct folding may not occur. One progress of the purification. If the properties are
effective way to increase solubility of foreign pro- unknown then a standard protocol of ion exchange
teins in E. coli is by coproduction of the bacterial (IEX), hydrophobic interaction (HIC), and gel filtra-
chaperones GroESL (Yasukawa et al., 1995). Copro- tion (GF) is followed. The three essential phases of
duction of GroESL with transcription factors and any purification are: capture, followed by interme-
oncogene products resulted in soluble protein. A diate purification to remove the bulk of impurities