Question

18. A DNA-binding protein is needed to be expressed and purified for structural studies. It has the following properties: (a) It is a human protein; (b) The overall molecular mass is about 150 kDa; (c) It contains rare codons; (d) It has multiple charged and hydrophobic amino acid residues; (e) Its pI is 5.4; It is sensitive to proteolytic degradation. Starting from the design for protein expression using an appropriate expression system, briefly describe how you would proceed logically to purify this protein to homogeneity (pure form). You may describe your answer with the help of a flow-chart if necessary. (13 marks)

Answer 1

DNA-protein is a class of diverse protein which binds to to DNA and can perform many functions like DNA/RNA polymerase, trancriptase activity, repressor, etc. Due to their DNA binding capability they can be purified using those domains as ligands for purification. Eg. Heparin is a  highly sufonated glycosaminoglycan which binds to large range of biomolecules as DNA binding protein as initiation factor, elongation factors, enzymes, growth factors, lipoproteins, etc. There are many techniques for purification which can be used to purify proteins from a mixture of protein. Eg. chromatography.

Chromatography is a physical method of separation and purification of compounds. this tchnique can be divided into two categories on physical basis by whioch stationary and mobile phase interact with each other and hence, classified as planar and column chromatography. Column chromatography is tube with stationary phase in it. column chromatography is further divided into adsorption and partition chromatography, size exclusion chromatography, ion exchange chromatography and affinity chromatography.

Affinity chromatography is widely used to purify proteins witrh affinity to some particular components as DNA binding proteins have affinity to DNA. In DNA binding affinity chromatography purification of specific specific DNA binding proteins. Before purification, agarose beads (insoluble matrix) of stationary phase.are synthesized by linking to double stranded oligonucleotide. These beads are filled in column which binds to selective protein recognizing the DNA sequence.

Steps included are

• Total cell protein are passed through column with different DNA sequence.
• Protein binds to DNA are separated from different proteins. Most of the DNA binding proteins have weak affinity for bulk DNA and retained on column by ionic attractions. waste proteins can be washed off by different salt concentration. proteins bound to DNA stick to beads and are eluted at later stages by different salt concentration.

For protein expression, bacterial host cells plasmids like DH5$\alpha$, BL21DE3, etc. are transfaected with DNA binding protein because bacterial cells are easy to grow and express protein in its original state (homology).

• culture bacterial cells in liquid broth overnight at 37$\degree$C in incubator at shaking condition for proper aeration.
• after overnight culture, take 1 ml of culture in eppendorf and centrifuge it at 4000rpm for 3 minutes. Lyse pellet and extract proteins from recombinant cell.
• run this protein mixture through the column and collect all the elutes.
• after chromatography is done and all elutes are collected, run sample through electrophoresis gel using ladder.
• The DNA binding protein will show one band only at 150 kDa ladder and only one protein band is present because during chromatography all waste proteins are washed off early and protein bound to DNA are eluted later at high salt concentrations.