Caspases,Paracaspases, and Metacaspases Methods and Protocols

4

biochemical studies, these methods have populated a list of more

than 1,400 caspase substrates [ 11 , 12 ]. In most circumstances, the

relevancy of these proteolytic events has not been determined, and

yet again, the availability of caspases for in vitro assays will help to

validate and study these substrates.

Over the years, we have developed an expertise in the expres-

sion, purification, and characterization of caspases. This chapter

describes the basic protocols for caspase expression in E. coli as His-

tagged proteins, their purification on immobilized metal affinity

chromatography (IMAC) columns, and the in vitro characteriza-

tion of their enzymatic activity. Caveats, pitfalls, and remedies for

individual caspases are discussed, and a broader discussion of the

production of specific molecular forms, and some protein engineer-

ing approaches for these enzymes are also presented. We propose a

work flowchart allowing for the expression, purification, and char-

acterization of recombinant caspases within a 5-day period (Fig. 1 ).

The physiological environment of caspases is the cytosol of a

cell. In that respect, the osmolarity and reducing conditions found

in both E. coli and mammalian cells are similar. Furthermore, there

is no peptidase with similar functions or activity in E. coli, making

this host ideal for expressing caspases. Finally, none of the

posttranslational modifications of caspases that occur in mamma-

lian cells (e.g., phosphorylation, ubiquitination, sumoylation)

or

Full-length caspase-

Transformation in

E. coli Pre-cultures Expression (5 h)

End expression

Freeze at -80 °C

IMAC purification

Freeze at -80 °C Titration Caspase assays

Inclusion bodies

Start solubilization Caspase assays

DEAE purification

Start refolding

End solubilization

IMAC purification

End refolding

Titration

Fig. 1 Timeline of protocols. The basic expression protocol takes 3 days. Add an extra day if the time necessary
to produce the protein is long (>12 h). Either way, purification is performed on the fourth day. The IMAC purifi-
cation is quick (1 day), and the basic characterization also takes 1 day. Because full-length caspase-8 is
insoluble when expressed in E. coli, the protocol is longer and involves denaturation of proteins, IMAC purifica-
tion, an optional DEAE anion exchange chromatography, and a full day to refold the protein into an active
enzyme. Purified caspase-8 is characterized immediately following purification

Dave Boucher et al.

http://www.ebook3000.com