Constructs and Purification
The A1LS has been previously observed in peanut extracts[17]. The
sequence of the observed peptide with minor modifications was used to
generate the sequence referred to as A1LS in this paper, but whose
formal designation is Ara h 1.0101 (25-83) A25G. Constructs for JR2.1:
Jug r 2.0101 (1-57) M0 added , JR2.2: Jug r 2.0101 (67-111) Q67G N68M, ,
and JR2.3: Jug r 2.0101 116-161 D116D R117M were prepared by identifying
their constituent CxxxC pairs and extending the sequence 10-20 residues
on either side. All constructs were cloned into the pDest expression
system with an N-terminal Glutathione S-transferase (GST) affinity tag
separated from the main sequence by a tobacco etch virus (TEV) protease
cleavage site. Cells expressing these constructs were grown in 2x YT
media to an OD of ~0.8 at 37oC. To
produce uniformly 15N-labeled or13C-15N-labeled A1LS for NMR, cells
were grown overnight in 1 L Luria Broth (LB), harvested, and
subsequently transferred to M9 media with15NH4Cl and, where applicable,13C-glucose as the sole nitrogen and carbon sources
respectively. Expression was induced with 0.5 mM Isopropyl
β-d-1-thiogalactopyranoside (IPTG) at 16oC overnight.
A1LS was purified from the resulting cells using an immobilized
glutathione column, eluted with 10 mM reduced glutathione in pH 7.4 PBS,
and incubated with TEV protease overnight to cleave the GST tag. Correct
disulfide bond pairing was achieved by diluting the sample and adding
oxidized glutathione to a final concentration of 2 mM, and 0.5 mM
reduced and oxidized glutathione respectively [38] (redox buffer),
the efficacy of which was subsequently verified using1H-15N NMR and mass spec (S2), and
further confirmed over the course of 3D structure determination
described subsequently. Following 30 minute incubation the sample was
loaded onto a Superdex75 26/600, and eluted with PBS to remove the GST
tag. The J2LS fragments J2.1, J2.2, and J2.3 were purified in a similar
manner with the following modification: the GST tag was removed via
incubation with TEV protease in the presence of 2 mM DTT, the latter of
which was required to ensure complete cleavage for some constructs. The
buffers in the resulting samples were exchanged for PBS, incubated with
redox buffer, and purified via SEC as described above.
The full length (FL) J2LS as identified by Downs et al. [16]
but with the addition of an N-terminal start codon (Met) and without the
C-terminal Arg was cloned into the Pet9a vector. The protein was
expressed in BL21 cells as above. Cells were lysed and the resulting
soluble proteins were precipitated using 25%, 50%, and 75% ammonium
sulfate. The 75% precipitate fraction was isolated and resuspended in
Buffer A (50 mM Tris-HCL pH 8.3, 100 mM NaCl) and loaded onto a
Mono-S-Sephrose anion exchange chromatography column. FL JR2 was eluted
using a linear gradient of Buffer A and Buffer B (50 mM Tris-HCL pH 8.3,
500 mM NaCl). FL J2LS-containing fractions were incubated for 30 minutes
with 2 mM/0.5 mM of reduced/oxidized gluthathione to ensure proper
disulfide bond formation before being loaded onto a Superdex75 26/600
sizing column and eluted with PBS to yield the final purified protein.