Supplementary Material
Enhanced protein stability through minimally-invasive, direct, covalent and site-specific
immobilization
Mark T. Smith¥+, Jeffrey C. Wu§+, Chad T Varner¥, and Bradley C. Bundy¥
¥
§
Department of Chemical Engineering
Department of Molecular and Microbiology
Brigham Young University
Provo, Utah, 84602
CONTENTS
Figure S1. Crystal Structure of sfGFP-related Protein.
Figure S2. Aminoacyl-tRNA Synthetase Optimization .
Figure S3. Scintillation and Fluorescence Data for Washes after pPaGFP Immobilization.
Figure S4. Freeze-thaw Activity for Free pPaGFP in Copper-containing Solutions.
3 nm
3 nm
5 nm
Figure S1. Crystal Structure of sfGFP-related Protein.
The potential implications of the residue location for unnatural amino acid incorporation were
investigated using the known crystal structure of a superfolder green fluorescent protein variant
closely related to the pPaGFP (pdbID 2B3P).1 In the Fig. S1, the beta-barrel (dark green) is
approximated as a cylinder with the loop of interest (pink) extending from the main body. The
pink loop contains the location for the unnatural amino acid incorporation (black).
Fluorescence (a.u.)
80
70
60
50
40
30
20
10
0
12
6
1
0
0
6
12
Magnesium Glutamate [mM]
Figure S2. Aminoacyl-tRNA Synthetase Optimization
Cell-free protein synthesis allows for direct access to the synthesis environment, enabling
optimization and maximization of synthesis cofactors. To maximize the unnatural amino acid
incorporation, the effect of different concentrations of aminoacyl-tRNA synthetase and
magnesium glutamate on the production of active pPaGFP was assessed.
800
700
500
400
300
200
Wash 2
Wash 3
Beads
5000
Fluorescence (a.u.)
600
Counts/min
6000
Wash 2
Wash 3
Beads
4000
3000
2000
1000
100
0
0
CuSO4
CuTet
Ctrl
CuSO4
CuTet
Ctrl
Figure S3. Scintillation and Fluorescence Data for Washes after pPaGFP Immobilization.
To ensure that the proteins were not remaining in solution or non-specifically binding to the
magnetic beads, the beads were washed 3 times using a PBS-Tween buffer. Each wash consisted
of isolating the magnetic beads, removing the supernatant, and resuspending the beads in 100 μL
PBS-Tween buffer. The final suspension (labeled “Beads”) was in 100 μL PBS and was vortexed
directly prior to analysis for a uniform suspension of beads. Displayed above are the results from
liquid scintillation and fluorescence analysis of supernantant from washes 2 and 3, along with the
final suspension containing beads. During the washing process, the unclicked pPaGFP was
removed to background or statistically insignificant levels. For the control reactions containing
no copper, the resuspended beads contained no or insignificant levels of pPaGFP. The prewash
and wash 1 results were removed to provide appropriate scaling. Error bars = standard deviation,
n=2.
ΔFluorescence (a.u.) / mol
pPaGFP
2
CuSO4
2
0
0
-2
-2
-4
-4
-6
-6
-8
-8
-10
-10
-12
Cu(I)Tet
-12
Read 1 Freeze Freeze Freeze Freeze
Thaw 1 Thaw 2 Thaw 3 Thaw 4
Read 1 Freeze Freeze Freeze Freeze
Thaw 1 Thaw 2 Thaw 3 Thaw 4
Figure S4. Freeze-thaw Activity for Free pPaGFP in Copper-containing Solutions.
Free pPaGFP was incubated under the same conditions as bead-immobilized pPaGFP and then
was left in its respective reaction solution. The unbound pPaGFP was subjected to multiple
freeze-thaw cycles (squares) or incubated at 4 oC (diamonds) and assayed for activity. Each
freeze-thaw cycle consisted of an incubation for 20 min at -80 oC followed by a 20 min
incubation at room temperature. Samples left unfrozen were maintained at 4 oC for 40 min
between assays. The results suggest that incubation with copper plays little role in terms of
stability during freeze-thaw cycles. Error bars = standard deviation, n=2.
References
(1)
Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and
characterization of a superfolder green fluorescent protein. Nat Biotech 2006; 24: 79-88.