Page 73 - 72_04

VOL. 72 (4), 611-627, 2006 HEME RESPONSIVENESS IN VITRO IS A COMMON...

MATERIAL AND METHODS

HRI Mutagenesis and Plasmid Construction

All HRI expression constructs were generated from pRSETB-
MHRI plasmid, which harbors wild type (wt) mHRI (11). Single
point mutations C409A and C550A were made by using the
QuickChangeTM site-directed mutagenesis kit (Strategene) as
described by the manufacturer, using the mutagenic primers shown
in Table I. HRI ?8 and HRI ?8/C550A were generated from pRSETB-
MHRI and pRSETB-MHRI/C550A plasmids, respectively, by
substitution of the cDNA fragment encoding aa 1 to 413 with a PCR
generated fragment encoding aa 1 to 405. The resultant plasmids,
pRSETB-MHRI ?8 and pRSETB-MHRI ?8/C550A, encode HRI
proteins lacking the 406 to 413 aa residues alone, or together with
the C550A point mutation. To generate the HRI ?137/C409A/C550A
mutant, the pRSETB-MHRI C409A/C550A plasmid was cut with
BamHI, yielding three fragments. The fragment corresponding to the
HRI central region (aa 138-501) was subcloned to produce a plasmid
encoding an HRI product that lacks the 1 to 137 aa residues and
contains the C409A and C550A mutations. HRI 138-580/C409A/C550A
was generated by substituting the Gln-581 CAA codon for a stop
codon (UAA) in the pRSETB-MHRI ?137/C409A/C550A plasmid.

HRI Q4 chimera was obtained as follows: the whole pRSETB-
MHRI plasmid without the region corresponding to the HRI kinase
insert domain (residues 235-389) (11) was amplified by PCR,
introducing restriction sites at both ends. The kinase insert domain
(residues 656-807) of GCN2 (5) was also amplified by PCR
introducing the same restriction sites. Both generated DNA
fragments were ligated to produce a pRSETB-MHRI Q4 vector
encoding an HRI protein with the MGCN2 insert sequence.

Prokaryotic expression of HRI proteins

The pRSETB-MHRI-wt and the pRSETB-MHRI-mutant plasmids
were used to transform a competent BL21 (DE3) pLys S strain of
E. coli. Protein expression and bacterial lysis were done as previously

615