Recombinant Sortase A5 protein

Catalog No: 13100 Format: 50 µg $450 Buy
Catalog No: 13101 Format: 250 µg $1,250 Buy

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Expressed In: E. coli Protein Species: S. aureus

Contents

A representative Technical Data Sheet (TDS) is provided here. Please refer to your lot-specific TDS upon arrival for specific buffer contents and lot-specific protein concentration.

Background

Sortase belongs to a class of transpeptidases that utilize an active site cysteine thiol to modify proteins by recognizing and cleaving a carboxy-terminal sorting signal, LPXTG (where X is any amino acid), between the threonine and glycine residues. Sortase A5 is an engineered pentamutant variant of the wild-type sortase from Staphylococcus aureus that is significantly more active than the wild-type sortase. Sortase A5 site-specifically labels antibodies or proteins when the LPXTG recognition sequence is displayed. Easily attach a wide variety of labels such as peptides, DNA, carbohydrates or fluorophores containing a poly-Glycine sequence (Gly)n (where n = 3 or more Glycine residues). Sortase A5 Pentamutant is covered by US patent number 9,267,127.

Application Notes

Sortase A5 recognizes an antibody or protein genetically engineered to contain the LPXTG motif (where X is any amino acid). Sortase A5 cleaves this sequence between the threonine and glycine residues and the terminal glycine is then replaced with any poly-Glycine (G)n label. Sortase A5 is used in Active Motif’s Sortag-IT™ Labeling Kits to attach HRP, biotin, fluorophores and other labels directly to Active Motif’s AbFlex™ recombinant antibodies (rAb). The activity of both wild type and Sortase A5 pentamutant proteins are Ca2+ dependent, therefore, Active Motif’s Sortag-IT labeling buffers and reagents are formulated to contain Ca2+ for optimal protein labeling. In addition, our AbFlex recombinant antibodies are provided in HEPES buffer, which is does not bind Ca2+ and will not interfere with the Sortase A5 enzymatic activity.

Protein Details

Recombinant Sortase A5 protein (S. aureaus, Uniprot A0A077UNB8-1), containing amino acid substitutions P94R, D160N, D165A, K190E and K196T, was expressed in E. coli and includes a C-terminal 6xHis Tag. The molecular weight of the protein is 17.8 kDa.

Protein Sequence: MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFA EENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPT
AVGVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLEHHHHHH

 

Recombinant Sortase A5 protein specificity for LPETG sequence.
Sortase A5 was incubated with LPETG, LAETG or LPESG substrates in reaction buffer, followed by fluorescence detection upon cleavage. Results demonstrate the high specificity of Sortase A5 for LPETG, minimal activity for substrate LPESGK, and no activity towards the mutated substrate LAETG.

Sortase A5 has increased labeling efficiency compared to the Wild-Type sortase.  The H3K9Ac AbFlex antibody (67 µg) was labeled with 5 µg Biotin (1.28 mM) using 1 µg Sortase A5 or Wild-Type sortase at 30 °C for 2 hr. with shaking. Top Panel: Following purification, 0.25 and 0.125 µg of labeled antibodies were run on an SDS-PAGE PAGE gel and labeling was detected using streptavidin-HRP.   Lanes 1 & 2: 0.25 µg and 0.125 µg of H3K9Ac AbFlex Ab labeled with wild-type Sortase, respectively. Lanes 3 & 4: 0.25 µg and 0.125 µg of H3K9Ac AbFlex Ab labeled with Sortase A5, respectively.
Bottom panel: The intensity of the labeling was quantified using BioRad’s Gel Doc imaging system, represented here as arbitrary units.

Storage

Store at -80°C to prevent degradation and avoid repeated freeze/thaw cycles. This product is guaranteed for 6 months from date of arrival.