Caspases,Paracaspases, and Metacaspases Methods and Protocols

205

binding studies were used to identify the calcium binding site,

study potential TbMCA2 substrates, and identify Z-VRPR-FMK

as a TbMCA2 inhibitor [ 17 ]. These results collectively gave new

insights into the mode of activation and substrate specifi city of

metacaspases and in particular of TbMCA2.

This chapter presents a detailed overview of the methods used

in our laboratory to overexpress, purify, and crystallize recombi-

nant TbMCA2 C213A. It includes details on crystal improvement

using seeding techniques and on obtaining a heavy atom derivative

using samarium in order to investigate the structure of the calcium-

binding site. In addition, it describes the use of fl uorogenic sub-

strate enzyme assays to calculate the activity of TbMCA2 and/or

the K (^) m of a chosen substrate. It also describes how to perform
inhibitor-binding assays and calculate the IC 50 for inhibitors. Many
of the methods presented here will be useful when working with
metacaspases from other species and for Clan CD cysteine pepti-
dases in general.
2 Materials
Expression plasmids for this work were originally produced using
the TbMCA2 coding sequence, amplifi ed from T. brucei genomic
DNA, inserted into the Nde1 and XhoI sites of pET28a+ (Novagen).
This produced an enzyme with an N-terminal His-tag and throm-
bin cleavage site, the latter of which was subsequently mutated to
remove the cleavage site arginine residue [ 10 ] ( see Note 1 ). All
mutations to TbMCA2 were constructed from this plasmid
(pGL1573) using the QuickChange Site-Directed Mutagenesis
Kit (Stratagene) and the appropriate oligonucleotide primers [ 17 ].
All solutions should be prepared with ultrapure water and
stored at room temperature unless otherwise stated. In general, all
chemicals should be of ACS reagent grade of 99 % purity, prepared
in double distilled water (ddH 2 O), and sterile fi ltered before use.

1. Expression vector for protein expression containing the meta-
   caspase genes and an N-terminal His-tag for protein purifi ca-
   tion ( see Note 2 ).


2. Competent cells, E. coli BL21 (DE3) or Rosetta (DE3) (e.g.,
   One Shot BL21 (DE3) chemically competent E. coli ,
   Invitrogen; Rosetta 2 (DE3) Singles, Millipore) ( see Note 3 ).


3. Magnetic stirrer and stirring bar.


4. Sterile bacterial cell spreaders.


5. Water bath set at 42 °C and a shaking incubator set at 37 °C.


6. Kanamycin stock (1,000×, 25 mg/mL) prepared in ddH 2 O,
   sterile fi ltered, and stored at −20 °C ( see Note 4 ).

2.1 Transformation
of Cells and Protein
Expression

Trypanosoma Metacaspases