Difference between revisions of "Part:BBa K5136229"

Revision as of 16:24, 1 October 2024

I0500-B0034-gst-linker-crs5-B0015

Biology

GST tag

Glutathione-S-transferase (GST) tag is a peptide tag derived from Schistosoma japonicum. GST tag has a large relative molecular mass of about 26 KDa and is often inserted at the N-terminus of target proteins. It facilitates the separation of target proteins from cell extracts by its affinity for glutathione In addition, most of these fusion proteins are stable and water-soluble (1).

CRS5

The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular protein and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn²⁺, Ni²⁺, Pb²⁺, Hg²⁺, Cd²⁺, as well as As³⁺. CRS5 is a metallothionein from S. cerevisiae. It is mainly responsible for copper ion homeostasis and detoxification in yeast cells (1).

Usage and Design

We decided to use MTs to treat wastewaters. In order to increase the stability of MTs, we added a GST tag to its N-terminal. Thus, this composite part BBa_K5136229 was constructed to express fused protein GST-linker-CRS5.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136229) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2584 bp) can be observed at the position between 2000 bp and 3000 bp (Figure 1).

Reference

1.D. B. Smith et al., Mr 26,000 Antigen of Schistosoma Japonicum Recognized by Resistant WEHI 129/J Mice is a Parasite Glutathione S-Transferase. Proc. Natl. Acad. Sci. U. S. A. 83, 8703-8707 (1986).
2.V. C. Culotta, W. R. Howard, X. F. Liu, CRS5 Encodes a Metallothionein-Like Protein in Saccharomyces Cerevisiae. J. Biol. Chem. 269, 25295-25302 (1994).

Sequence and Features