Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

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.
3. Select and copy the 45 nucleotide reverse translation of the peptide sequence.
4. Download the GenBank File for pBAD-BirA-e....

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 variants^7,8. This ensures that rare clones are selected and that non-biotinylated bac.......

Materials

Name	Company	Catalog Number	Comments
10% precast polyacrylamide gel	Bio-Rad	4561033
Ampicilin	Sigma-Aldrich	A1593
ApE - A plasmid editor v2.0	NA	NA	downloaded from http://jorgensen.biology.utah.edu/wayned/ape/
Arabinose	Sigma-Aldrich	A3256
Biotin	Sigma-Aldrich	B4501
DMSO	Sigma-Aldrich	D2650
DPBS (10X), no calcium, no magnesium	ThermoFischer Scientific	14200083
DpnI restriction enzyme	New England BioLabs	R0176
Dynabeads MyOne Streptavidin C1	ThermoFischer Scientific	65001
GenElute Plasmid Miniprep Kit	Sigma-Aldrich	PLN350
GeneMorph II EZClone Domain Mutagensis kit	Agilent Technologies	200552
Glucose	Sigma-Aldrich	G8270
Glycerol	Sigma-Aldrich	G5516
Immobilon-P PVDF Membrane	Millipore	IPVH15150
IPTG	Sigma-Aldrich	I6758
LS Columns	Miltenyi Biotec	130-042-401
NaCl	Sigma-Aldrich	S7653
NEB 5-alpha Competent E. coli	New England BioLabs	C2987
NuPAGE LDS Sample Buffer (4X)	ThermoFischer Scientific	NP0007
NuPAGE Sample Reducing Agent (10X)	ThermoFischer Scientific	NP0009
pBAD-BirA-eCPX-AP	Addgene	121907	Used a template and positive control
pBAD-BirA-eCPX-AP(K10A)	Addgene	121908	negative control
Q5 High-Fidelity DNA Polymerase	New England BioLabs	M0491	For insertion of peptide sequence in pBAD-BirA-eCPX-AP, any high fidelity polymerase will do
QuadroMACS Separator	Miltenyi Biotec	130-090-976
Skim Milk Powder	Sigma-Aldrich	70166
Streptavidin-HRP	Agilent Technologies	P0397
T7 Express lysY/Iq Competent E. coli	New England BioLabs	C3013
Tryptone	Millipore	T9410
Tween-20	Sigma-Aldrich	P9416
Western Lightning Plus-ECL	PerkinElmer	NEL103001EA
Yeast extract	Sigma-Aldrich	Y1625