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In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1

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Abstract

We present the in vivo biosynthesis of wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells using an intramolecular native chemical ligation in combination with a modified protein splicing unit. SFTI-1 is a small backbone cyclized polypeptide with a single disulfide bridge. A small library containing multiple Ala mutants was also biosynthesized and its activity was assayed using a trypsin-binding assay. This study clearly demonstrates the exciting possibility of generating large cyclic peptide libraries in live E. coli cells, and is a critical first step for developing in vivo screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold.

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Abbreviations

AMC:

7-Amido-4-methyl-coumarin

CBD:

Chitin-binding domain

Cbz:

Benzyloxycarbonyl

CCK:

Cyclic cystine-knot motif

BBI:

Bowman–Birk inhibitor

EDTA:

Ethylenediaminetetraacetic acid

EtSH:

Ethanethiol

GdmCl:

Guanidinium hydrochloride

GSH:

Glutathione

HPLC:

High performance liquid chromatography

LB:

Luria–Bertani

MAP:

Methionyl aminopeptidase

MESNA:

Mercaptoethanesulfonic acid

NHS:

N-hydroxysuccinimide ester

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethylsulfonyl fluoride

SDS:

Sodium dodecyl sulfate

SFTI-1:

Sunflower trypsin inhibitor 1

TFA:

Trifluoroacetic acid

UV-Vis:

Ultraviolet-visible

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Acknowledgments

Work was supported by funding from the School of Pharmacy at the University of Southern California and Lawrence Livermore National Laboratory.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences and Pharmacology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, PSC 616, Los Angeles, CA, 90033, USA

    Julio A. Camarero

  2. Lawrence Livermore National Laboratory, Livermore, CA, 94550, USA

    Jeffrey Austin, Richard H. Kimura, Youn-Hi Woo & Julio A. Camarero

Authors
  1. Jeffrey Austin
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  2. Richard H. Kimura
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  3. Youn-Hi Woo
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  4. Julio A. Camarero
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Correspondence to Julio A. Camarero.

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Austin, J., Kimura, R.H., Woo, YH. et al. In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1. Amino Acids 38, 1313–1322 (2010). https://doi.org/10.1007/s00726-009-0338-4

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