Question

How would you puriry a protein from inside a bateria cell. How might you lyse bacteria? and remove the cellular debris?

Answer 1

Ans:- protein can be purified using one of the following method given in step 4 purification of protein. cell lysis can be done by method given in step 1 extraction of protein from cell. step 2 and 3 shows how to remove cellular debris from cell lyse mixture.

protein purification include following steps, 1. extraction of protein from cell 2. precipitation of protein 3. ultracentrifugation of protein mixture 4. purification

1. extraction of protein from cell:- protein from inside cell need to be extracted by lysing cell membrane. this can be done by one of following method i) repeated freezing and thawing of cell ii). homogenization by high pressure iii). sonification iv). by detergent or by lysosomes. once cell is lysed proteins and debris can be separated by centrifugation. cell debris form a pellet at the bottom of tube and protein will remain in supernatant liquid.

2. precipitation:- supernatant liquid contains other soluble compound so proteins can be separated by adding ammonium sulfate which makes proteins precipitation.

3. ultracentrifugation:- precipitated protein can be separated from other soluble compound by ultracentrifugation. precipitated protein would form pellet at the bottom of centrifugation tube and other soluble compound will remain in supernatant liquid, supernatant can be discarded and pellet is then taken to further purification steps.

4. purification:- proteins in pellet can be separated by one of following method i). size exclusion chromatography ii). ion exchange chromatography iii). affinity chromatography iv). HPLC. lets take size exclusion chromatography to understand protein purification. principle of size exclusion is separation is based on size of protein particles. protein mixture to be separated pour into column with eluent. column is filled with gel which act as a stationary phase. eluent is mobile phase. smaller proteins get trapped into gel and hence move slower through column, get eluted latter. bigger proteins do not get trapped in the gel move faster through gel and get separated early.