4. Cadherins are calcium-dependent cell adhesion proteins. Your supervisor asks

Question

4. Cadherins are calcium-dependent cell adhesion proteins. Your supervisor asks you to devise a plan to recombinantly express and purify the cytoplasmic domain of human E- cadherin in E. coli. Using the topology table from UniProt below as a guide, outline briefly what you would do experimentally to achieve this. Uniprot P12830 (CADH1_Human) Feature key Position (amino acids)Description Topological domain155 - 709 Transmembrane Topological domain731 -882 Extracellular Helical Cytoplasmic 710-730

Explanation / Answer

Using uniprot we can find out the amino acid sequence of cytoplasmic domain and its mRNA sequence this we can covert into cDNA sequence. To express the recombinant protein , we clone cDNA sequence of E cadherin cytoplasmic domain in expression vector, plasmid based expfession vector we can use to express eukaryotic protein. Now, our host cell is E.Coli, BL21(DE3) strain used that overcome the effect of codon bias. After the cloning, small scale test expression done , it is predictive tool to determine which of the derivatives clones actually producing a recombinant protein. It also help in establishment of optimum scale for large scale growth. Then, purification process involves bacterial cell lysis and extraction of fraction of recombinant protein without unwanted proteolysis, protein oxidation and contamination with genomic DNA. Than , this fraction pass through the chromatography coumn to purify recombinant protein.

Therefore, uniprot provide information of position of amino acid of cytoplasmic domain , which further convert into desired mRNA sequence and ultimately cloned into E.coli and purification.

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