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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

Abstract

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Introduction

Biotinylation of a protein creates a powerful tag for its affinity isolation and detection. Enzymatic protein biotinylation is a highly specific post-translational modification catalyzed by biotin-protein ligases. The E. coli biotin-protein ligase BirA is extremely specific and covalently biotinylates only a restricted number of naturally occurring proteins at specific lysine residues1. The advantages of the BirA catalyzed biotinylation are currently harnessed by fusing the target protein with a small synthetic 15-amino-acid biotin acceptor peptide (AP) that is effectively biotinylated2 and allows for the highly....

Protocol

1. Insertion of Peptide Coding Sequencing Sequence in pBAD BirA-eCPX-AP

NOTE: To select for BirA variants that biotinylate a native target protein, start by identifying a 15-amino acid peptide sequence in the proteins primary sequence that contains at least one lysine (K) residue.

  1. Go to the sequence manipulation suite12.
  2. Paste the identified 15 amino acid peptide sequence into the input box in FASTA format and press Submit<.......

Representative Results

Western blot of pBAD-BirA-eCPX-AP expressing bacteria produces a ~22 kDa streptavidin-reacting band consistent with the molecular weight of eCPX (Figure 2a). Unlike BirA-6xHis, biotinylated eCPX-AP was present in both uninduced and induced cultures (Figure 2a) due to a small degree of T7 promoter activity even in uninduced cultures and subsequent biotinylation of the AP by endogenous BirA. In BirA-eCPX-AP(K10A) expressing cultur.......

Discussion

As for all selection methods, the stringency of the washing steps is of utmost importance. Since bacteria do not need to be eluted from the beads before the amplification of the selected clones, the high affinity binding between biotin and streptavidin can be used instead of using lower affinity avidins, as previously done with the phage display system, for the selection of BirA variants7,8. This ensures that rare clones are selected and that non-biotinylated bac.......

Acknowledgements

The authors thank Mohamed Abdullahi Ahmed for the expert technician assistance. This work was supported by grants from the Lundbeck Foundation, the Novo Nordisk Foundation, the Danish Kidney Association, the Aase og Ejnar Danielsen Foundation, the A.P. Møller Foundation for the Advancement of Medical Science, and Knud and Edith Eriksen Memorial Foundation.

....

Materials

NameCompanyCatalog NumberComments
10% precast polyacrylamide gelBio-Rad4561033
AmpicilinSigma-AldrichA1593
ApE - A plasmid editor v2.0NANAdownloaded from http://jorgensen.biology.utah.edu/wayned/ape/
ArabinoseSigma-AldrichA3256
BiotinSigma-AldrichB4501
DMSOSigma-AldrichD2650
DPBS (10X), no calcium, no magnesiumThermoFischer Scientific14200083
DpnI restriction enzymeNew England BioLabsR0176
Dynabeads MyOne Streptavidin C1ThermoFischer Scientific65001
GenElute Plasmid Miniprep KitSigma-AldrichPLN350
GeneMorph II EZClone Domain Mutagensis kitAgilent Technologies200552
GlucoseSigma-AldrichG8270
GlycerolSigma-AldrichG5516
Immobilon-P PVDF MembraneMilliporeIPVH15150
IPTGSigma-AldrichI6758
LS ColumnsMiltenyi Biotec130-042-401
NaClSigma-AldrichS7653
NEB 5-alpha Competent E. coliNew England BioLabsC2987
NuPAGE LDS Sample Buffer (4X)ThermoFischer ScientificNP0007
NuPAGE Sample Reducing Agent (10X)ThermoFischer ScientificNP0009
pBAD-BirA-eCPX-APAddgene121907Used a template and positive control
pBAD-BirA-eCPX-AP(K10A)Addgene121908negative control
Q5 High-Fidelity DNA PolymeraseNew England BioLabsM0491For insertion of peptide sequence in pBAD-BirA-eCPX-AP, any high fidelity polymerase will do
QuadroMACS SeparatorMiltenyi Biotec130-090-976
Skim Milk PowderSigma-Aldrich70166
Streptavidin-HRPAgilent TechnologiesP0397
T7 Express lysY/Iq Competent E. coliNew England BioLabsC3013
TryptoneMilliporeT9410
Tween-20Sigma-AldrichP9416
Western Lightning Plus-ECLPerkinElmerNEL103001EA
Yeast extractSigma-AldrichY1625

References

  1. Polyak, S. W., Abell, A. D., Wilce, M. C. J., Zhang, L., Booker, G. W. Structure, function and selective inhibition of bacterial acetyl-CoA carboxylase. Applied Microbiology and Biotechnology. 93 (3), 983-992 (2011).
  2. Schatz, P. J.

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