Supplemental Figure 1. Native PAGE gel electrophoresis of cell-free expressed NLPs.
The cell free reactions are setup with 1μg/mL pIVEX-2.4d-∆49ApoA1 and 2mg/mL lipid
(Lane 1: 90 % DMPC and 10% PEG 5k -CA
8
, molar ratio; Lane 2: 100% DMPC), BODIPY-FL,
30C at 990rpm for 4 hours. All samples were loaded along with a NativeMark Protein
Standard (Lane M). The pictures were taken with GE-TYPHOON 9410 using laser/filter
488nm/520nm.
M 1 2
480kDa
242kDa
66kDa

62kDa
49kDa
38kDa
28kDa
17kDa
14kDa
Supplemental Figure 2. Telodendrimer addition increases the yield and solubility of
nanoparticles. A total of 1 ml cell-free expression reactions were run with and without
Telodendrimer PEG 5K -CA
8
in DMPC. A typical comparison of the differing fractions produced throughout the purification is shown. The following symbols are M = Molecular
Weight Marker, T = total reaction, FT = flow through and E = elution fractions.
M T
Δ49ApoA1 w/ DMPC
FT E
Δ49ApoA1 w/
1% PEG
5K
-CA
8 in DMPC
T FT E
188KDa
98KDa
∆49ApoA1

Supplemental Figure 3. The size distribution of TD-lipid particles measured by
Dynamic Light Scattering. TD-lipid particles were produced with distinct size characteristics depending on the type of telodendrimer mixed with DMPC. TD-lipid particles were bigger than corresponding TD-NLPs. (Figure 2 in the article).
140
120
100
80
60
40
20
0
1%
10%
PEG2K-CA4
30,4
18,83
PEG2K-CA8
51,7
93,3
PEG5K-CF4
44,4
49,3
PEG5K-CA8
79,7
103,25

Supplemental Figure 4. The size distribution of TD-NLPs measured by Dynamic Light
Scattering. Increase in the amount of telodendrimer to lipid ratio was associated with greater level of aggregation.
120,00%
96,30%
100,00%
77,60% 78,20%
80,00%
60,00%
40,00%
20,00%
0,00%
14,20%
20,50%

Supplemental Figure 5. Size exclusion chromatography (SEC) trace of PEG 5K -CA
8
TD-
NLP and pre-isolated, monodispersed normal NLP. TD-NLP was pre-purified with nickel affinity chromatography. TD-lipids were not identified as a distinct peak in the SEC experiments following affinity chromatography. When SEC was run prior to affinity chromatography for the TD-lipids, they were normally found in the void volume (data not shown). Normal NLP’s were also purified using SEC. TBS was used as the running buffer. All
NLP/TD-NLP appeared as a single peak indicates that they were monodispersed in size.
The size of TD-NLP appeared to be larger than normal NLP, consistent with DLS results.
Free protein peaks were observed as was small molecules probably present in the buffer.

Supplemental Figure 6. Cell–free BR expression with presence of TD-lipid result in
production of functional yet insoluble membrane protein. The cell free reactions are setup with 10 μg/mL pIVEX-2.4d-boP and 2 mg/mL lipid (100% DMPC or 99.5% DMPC and
0.5% TD, molar ratio, etc) and 50μM all trans retinal, 30  C at 990rpm for 4 hours. (A) The pictures of the tubes were taken after the reaction was complete. (B) Tubes were centrifuged at 14,000 rpm for 10 mins and visually checked for solubility. The functional
BR protein was seen in the pellet. The tubes are as follows: (1) Cell-free Lysate without
DNA, (2) Cell-free expression of BR with DMPC, (3) Cell-free expression of BR with DMPC and 0.5% telodendrimer PEG 5k -CA
8
, (4) Cell-free expression of BR with DMPC and 1% telodendrimer PEG 5k -CA
8
, (5) Cell-free expression of BR with DMPC and 2% telodendrimer
PEG 5k -CA
8
, (6) Cell-free expression of BR with DMPC and 5% telodendrimer PEG 5k -CA
8
, (7)
Cell-free expression of BR with DMPC and 10% telodendrimer PEG 5k -CA
8
.