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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.
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.
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....
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.
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.......
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.......
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.
....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 |
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