Basic Information:
CHST11 (N-acetylgalactosamine sulfate transferase 11) is an enzyme associated with glycan modification and plays a key role in the process of glycan synthesis. The CHST11 enzyme converts N-acetylgalactosamine to N-acetylgalactosamine sulfate, which is involved in the production of sulfacid modifications in the cornea and cartilage matrix. Understanding the function and mechanism of CHST11 is of great significance for the study of drug development in corneal and cartilage diseases and related diseases. The experimental design was designed to express and purify CHST11 recombinant protein and to analyze its enzymatic activity.
Experimental design:1Clone the CHST11 gene: Collect mRNA from target tissues (e.g., human cornea or cartilage tissue) and synthesize the cDNA of the CHST11 gene using RT-PCR.
Catalog No. PA1000-5542
2.Subcloning of CHST11 gene: Subclone CHST11 cDNA into a suitable expression vector (e.g., PET28A) to construct a recombinant protein expression plasmid.
3.Transform and express proteins: Recombinant plasmids are transformed into E. coli expression host strains (e.g., BL21) and expressed proteins are induced by IPTG.
4.Extraction of recombinant proteins: Cells are lysed by cell disruption by sonication or freeze-thaw to dissolve the target protein in lysis buffer.
5.Protein purification: Purify the protein of interest using the His-tag using a Ni2+ column or other affinity purification column.
6.Enzyme activity assay: A known enzyme activity assay was used to detect the enzymatic activity of CHST11 recombinant protein under suitable conditions. For example, CHST11 enzyme activity is analyzed by measuring the rate of acetylgalactosamine sulfation.
7.Results: The comparison of CHST11 enzyme activity was calculated, and the results were compared and verified with the existing research results.
Through the above experimental procedures, we can successfully express, purify, and detect the enzymatic activity of the recombinant protein CHST11, thereby deepening the understanding of the function and mechanism of the protein, and providing an experimental basis for the research and drug development of related diseases.