Phage Display Evolution of a Peptide Substrate for Yeast Biotin Ligase and Application to Two-color Quantum Dot Labeling of Cell Surface Proteins

Overview

Journal J Am Chem Soc

Publisher American Chemical Society

Specialty Chemistry

Date 2007 May 3

PMID 17472384

Citations 22

Authors

Irwin Chen

Yoon-Aa Choi

Alice Y Ting

Affiliations

Soon will be listed here.

Abstract

Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

Citing Articles

Biotin protein ligase as you like it: Either extraordinarily specific or promiscuous protein biotinylation.

Cronan J Proteins. 2023; 92(4):435-448.

PMID: 37997490 PMC: 10932917. DOI: 10.1002/prot.26642.

Expanding the Chemical Diversity of Genetically Encoded Libraries.

Iskandar S, Haberman V, Bowers A ACS Comb Sci. 2020; 22(12):712-733.

PMID: 33167616 PMC: 8284915. DOI: 10.1021/acscombsci.0c00179.

Molecular Sensing with Host Systems for Hyperpolarized Xe.

Jayapaul J, Schroder L Molecules. 2020; 25(20).

PMID: 33050669 PMC: 7587211. DOI: 10.3390/molecules25204627.

A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity.

Granhoj J, Dimke H, Svenningsen P Sci Rep. 2019; 9(1):4118.

PMID: 30858523 PMC: 6411976. DOI: 10.1038/s41598-019-40984-x.

Directed evolution provides insight into conformational substrate sampling by SrtA.

Suliman M, Santosh V, Seegar T, Dalton A, Schultz K, Klug C PLoS One. 2017; 12(8):e0184271.

PMID: 28859178 PMC: 5578623. DOI: 10.1371/journal.pone.0184271.

References

Polyak S, Brautigan P, Wallace J . Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity. J Biol Chem. 1999; 274(46):32847-54. DOI: 10.1074/jbc.274.46.32847. View

Heinis C, Schmitt S, Kindermann M, Godin G, Johnsson K . Evolving the substrate specificity of O6-alkylguanine-DNA alkyltransferase through loop insertion for applications in molecular imaging. ACS Chem Biol. 2006; 1(9):575-84. DOI: 10.1021/cb6003146. View

Cull M, Schatz P . Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol. 2000; 326:430-40. DOI: 10.1016/s0076-6879(00)26068-0. View

Polyak S, MULHERN T, Cronan Jr J, Wallace J . Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains. J Biol Chem. 2000; 276(5):3037-45. DOI: 10.1074/jbc.M003968200. View

Deng S, Liu W, Simmons C, Moore J, Tian G . Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening. Comb Chem High Throughput Screen. 2001; 4(6):525-33. DOI: 10.2174/088800199276958. View

Stroffekova K, Proenza C, Beam K . The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Pflugers Arch. 2001; 442(6):859-66. DOI: 10.1007/s004240100619. View

Adams S, Campbell R, Gross L, Martin B, Walkup G, Yao Y . New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc. 2002; 124(21):6063-76. DOI: 10.1021/ja017687n. View

Athavankar S, Peterson B . Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast. Chem Biol. 2004; 10(12):1245-53. DOI: 10.1016/j.chembiol.2003.11.007. View

Kim H, Hoja U, Stolz J, Sauer G, Schweizer E . Identification of the tRNA-binding protein Arc1p as a novel target of in vivo biotinylation in Saccharomyces cerevisiae. J Biol Chem. 2004; 279(41):42445-52. DOI: 10.1074/jbc.M407137200. View

10.

Barker D, Campbell A . Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli. J Mol Biol. 1981; 146(4):469-92. DOI: 10.1016/0022-2836(81)90043-7. View

11.

Samols D, Thornton C, Murtif V, Kumar G, Haase F, Wood H . Evolutionary conservation among biotin enzymes. J Biol Chem. 1988; 263(14):6461-4. View

12.

Cronan Jr J . Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem. 1990; 265(18):10327-33. View

13.

Devlin J, Panganiban L, Devlin P . Random peptide libraries: a source of specific protein binding molecules. Science. 1990; 249(4967):404-6. DOI: 10.1126/science.2143033. View

14.

Matthews D, Wells J . Substrate phage: selection of protease substrates by monovalent phage display. Science. 1993; 260(5111):1113-7. DOI: 10.1126/science.8493554. View

15.

Schatz P . Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology (N Y). 1993; 11(10):1138-43. DOI: 10.1038/nbt1093-1138. View

16.

Bower S, Perkins J, Yocum R, Serror P, Sorokin A, Rahaim P . Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon. J Bacteriol. 1995; 177(9):2572-5. PMC: 176921. DOI: 10.1128/jb.177.9.2572-2575.1995. View

17.

Marini P, Li S, Gardiol D, Cronan Jr J, de Mendoza D . The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis. J Bacteriol. 1995; 177(23):7003-6. PMC: 177574. DOI: 10.1128/jb.177.23.7003-7006.1995. View

18.

Athappilly F, Hendrickson W . Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing. Structure. 1995; 3(12):1407-19. DOI: 10.1016/s0969-2126(01)00277-5. View

19.

Boder E, Wittrup K . Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997; 15(6):553-7. DOI: 10.1038/nbt0697-553. View

20.

Stolz J, Ludwig A, Sauer N . Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation. FEBS Lett. 1998; 440(1-2):213-7. DOI: 10.1016/s0014-5793(98)01454-9. View