Part:BBa_K2721027
LDH with SdyTag and SpyCatcher
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 43
Illegal AgeI site found at 79 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 976
Usage and Biology
The covalent nature of Tag-Catcher interaction has facilitated the processing of proteins into various materials forms including all-protein-based, chemically cross-linked hydrogels, functional layer-by-layer thin films, hybrid colloidal assemblies, and living materials[1]. The SpyTag/SpyCatcher, SdyTag/SdyCatcher system are convenient protein coupling tools for irreversible peptide-protein ligation. According to papers, Spytag forms a covalent isopeptide bound to its protein partner SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein[2]. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcher derived from a related fibronectin-binding protein in Streptococcus dysgalactiae[3].
Fig1 Structure of SpyTag/SpyCatcher.[2]
Considering that the tag-catcher pairs are ideal for binding and immobilization, our team use them as scaffold of protein assembly. LDH is one of the enzymes fixed on the scaffold to catalyze relevant chemical reaction.
Approach and Result
The part was assembled with T7 promoter, RBS and T7 terminator in pET26b+ plasmid vector. E. coli strain BL21(DE3). After growing to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We get the protein product via sonication. With N-terminal His-tag, it can be purified by Ni-beads. [4]
Fig2 SDS-PAGE and Western Blot | A, SDS-PAGE, Lane M1 is protein marker, Lane 1 is BSA (2.00 μg), Lane 2 is SpyCatcher-LDH-SdyTag. B, Western Blot, Lane M2 is protein marker, Lane 3 is SpyCatcher-LDH-SdyTag.
We used BSA Standard Curve to qualifity . The OD595 of SpyCatcher-LDH-SdyTag is 0.509, and its corresponding concentration is 2.40mg/ml.
Reference
1. J Fang, WB Zhang. Genetically Encoded Peptide-protein Reactive Pairs. Acta Polymerica Sinica 2018 Jan (4):429-444.
2. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7.
3.Tan LL, Hoon SS, Wong FT. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10): e0165074.
4. P Hengen. Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences, 1995,20(7): 285-286.
5. Rakitina D.V., Baikova J.P., Ladygina,V.G., Manolov,A.I., Kanygina,A.V. Investigation of E. coli samples isolated from patients with Crohn's disease.
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