Preparation of labeled cyclic peptides with PUREfrex.
〇Mikiko Nakamura,,Kumiko Tsuihiji, Shizue Katoh,,Kanehisa Kojoh
GeneFrontier Corporation (https://www.genefrontier.com/contact/)[Background]
We reported that various cyclic peptides were selected simply and effectively by
Ribosome display with PUREfrex (PUREfrexRD). But for preparation of thosepeptides for further investigation, it takes a lot of cost and time with chemical
synthesis. Therefore, we have developed a new preparation method of labeled
cyclic peptides in high throughput manner with PUREfrex.
[Methods]
・Cyclic peptides were expressed as the N-term Maltose Binding Protein (MBP)
fused cyclic peptide.
・Cyclic peptides were released by digesting with Enterokinase.
・Labeled puromycin was added to PUREfrex to generate the C-term labeled cyclicpeptides.
・Labeling efficiency of puromycin were investigated with FITC-puromycin under
severalconditions. (+/- Release factor mix (RF mix;RF1 ,2 ,3 and RRF), Removing the stopcodon from DNA template, PUREfrex Ver.1.0/2.0)
・Optimized conditions were applied for actual screening of cyclic peptides against
CTLA4.

(2) Preparation of labeled cyclic peptides withPUREfrex(3) Example; Peptide screening for cyclic peptide toCTLA4, derived from affinity maturation library(3)-1, Immune checkpoint via CTLA4Enterokinase digestion
FLAG CMBPC PMBPCFLAGC P

Cyclic peptide
CPC

Labeled-Puromycin(Expression Construct)
T7 promoter

MBP

FLAG

Stop codon★★
Cys

MycCys[Results]
PUREfrex RFmix(-) showed the best efficiency of FITC-puromycin uptaking at Cterminal of full length proteins. In contrast, in the case of using PUREfrex RFmix(+),
the translation reaction stopped in the middle of the protein by adding FITCpuromycin. But the absence of stop codon in DNA template improved the uptaking
efficiency of FITC-puromycin even under using PUREfrex RFmix(+) in some cases.
The application of Biotinylated-puromycin instead of FITC-puromycin led to higher
sensitive ELISA with Streptavidin HRP even in free peptide format.
In the screening of CTLA4 binders with this method, we were able to identify thebest cyclic peptide with good EC50/IC50.

(3)-2, Evaluation of amino acid sequence and ELISA in
MBP fusion or free peptide format after affinity
maturation by PUREfrexRD[Conclusion]
This new method for preparation of cyclic peptides with PUREfrex will contributefor simple and effective high throughput functional screening of cyclic peptides,
and will be applied to the development of peptide based drugs.

(1) Problems for developing cyclic peptide
The physical properties of cyclic peptide displayed by in vitro selection
technologies such as ribosome display or phage display, and differed from those insynthesized free peptide, for example, followings are major points for thedifference.

・Isoelectric Point
・Fluctuation
・Solubility

Peptide A (pI = 8.25)

Stop codon (-)

Stop codon (+)
RF mix (+)
1000

100

10

RF mix (-)
1000

100

10RF mix (+)
1000

Puromycin (µM)

100

Mutation : Every roundRF mix (-)

10

1000

100

10

Sub‑cloning
Sequencing

75 K

75 K

Clones from affinity
maturation library

MBP-fusion
peptide(1)-1, Effect of Isoelectric Point
Selection of cyclic peptides
by in vitro display technologies

Washing time
1st round : 2h
2nd round : 12h
3rd round : 72h(2)-1, Effect of RF mix, Stop codon, andConcentration of labeled-puromycin

MBP-Fusion Peptide

PeptideS/N ratio of peptide

25 K

25 K

Peptide A
Peptide B
Peptide C

Peptide B (pI = 3.66)(Conditions)
Translation system : PUREfrex 1.0 DS supplement, RF mix (-)/(+)
Time : 60 min
Temperature : 37℃℃
Volume : 10 µL / tube
Input template DNA : 50 fmol /10 µL
Labeling : FITC-puromycin

RF mix(-), Stop codon(-), and 100 µM labeled puromycin was the best.

(2)-2, Comparison of expression between PUREfrex
1.0 and 2.0Binder A Binder B

Detection: anti-FLAG M2 HRP

The binding activities of both cyclic peptides in ELISA showed that Isoelectric
Points of free cyclic peptides and pH of buffer solutions have great impact on its
activity, although both had same binding activity on in vitro display technology.

PUREfrex 1.0

2.0

1.0

2.075 K(1)-2, Effect of Peptide Fluctuation
MBP-fusion
peptideAnti-Fc HRP(Conditions)
RF mix : (-)
Time : 120 min
Temperature : 37℃℃
Volume : 10 µL / tube
Input template DNA : 50 fmol /10 µL
Stop codon : (-)
Labeling : FITC-puromycin (100 µM)

FcStreptavidin HRP

Target protein

PEG25 K

Cyclic peptide

Target protein

BiotinStreptavidin
ELISA①①
Bait: Biotinylated Cyclic Peptide
Analyte : Fc-fused Target protein
Detection : Anti-Fc HRP

Binders with good binding activity were selected in free peptide format.
Key AAs and their positions were identified.

(3)-3, Measurements of affinity (EC50) as MBP-fusion
peptidePUREfrex 2.0 was better than PUREfrex 1.0.
FcELISA②②
Bait: Fc-fused Target protein
Analyte : Biotinylated Cyclic Peptide
Detection : Streptavidin HRP(2)-3, Release of cyclic peptide by Enterokinase
treatment
12

Lane 1：：Enterokinase untreated
Lane 2：：Enterokinase treated

MBP-fusion peptide

Time : 60 min
Temperature : 37℃℃
Enterokinase : 0.5U/10 µLµg/ml

E.coli expression host : BL21(DE3)
Purification : His tag purification

Affinity matured binders were identified.
The affinities of the binder No. 19, 10 and 24 were improved
about 50 times higher than that of the original binder.

Cyclic peptide

The binding activity depended on immobilized format, which suggests molecularfluctuation have impact on its activity.

(1)-3, Effect of Solubility
・In vitro display technologies generated various binders
independent from hydrophobicity/hydrophilicity.
・Highly hydrophobic peptide was insoluble or slightly soluble
in water, and was hard to synthesize, or in some cases, notto be synthesized at all.

Labeled cyclic peptide was effectively released by Enterokinase.

(2)-4, ELISA in different format, MBP fusion peptide
and free peptide labeled with Biotin-Puromycin

Asterisk (★
★）indicate that binders were performed to chemicalsynthesis.

(3)-4, Measurements of inhibition (IC50) as syntheticpeptides
Anti-Fc-Ab HRP

12

Fc - CTLA4
Low affinity
Cyclic Peptide

50 K

High affinity
Cyclic Peptide

Screening in free peptide format is necessary.

Non-inhibition

37 KInhibition

But Chemical synthesis takes
high cost and a long time.

No.19 IC50 : 22µµM

SolutionsPreparation of free peptides with PUREfrex
・Possible to prepare various peptides with low cost in ashort time.
・Possible to label the synthesized peptide easily.

Binder No.19 showed good inhibitory activity against interaction between CD80 andCTLA4 in fully synthetic peptide format.

25 K
Lane 1：：Anti-FLAG-HRP
Lane 2：：Streptavidin-HRP

Streptavidin HRP

Target protein
Free cyclic peptide labeled with biotin was released and detected in ELISA.

It enables high throuhghput functional screening ofvarious binders in free peptide format

CD80

Applicable for high-throughput screening in free peptide format.

Summary
1, We established new preparation method for labeled cyclic peptide
with puromycin and optimized PUREfrex.
2, In the screening of CTLA4 binders by this method, wesucceeded to select lead cyclic peptides with good EC50/IC50 assynthetic peptide format.