Protein purification using intein-hydrophobin tag and alcohol precipitation

A method of purifying a target protein, the method comprising:

• (a) preparing a cell culture supernatant comprising a fusion protein, wherein the fusion protein comprising:
• (i) a target protein domain;
• (ii) a self-cleaving intein;
• (iii) at least one hydrophobin protein domain, wherein the intein is located between the target protein domain and the hydrophobin domain;
• (b) adding a C1-C3 alcohol to the cell culture supernatant making a first alcohol solution;
• (c) stirring the first alcohol solution forming a first stirred solution;
• (d) centrifuging the first stirred solution forming a first precipitate and a first supernatant, wherein the first supernatant comprising the fusion protein;
• (e) separating the first supernatant comprising the fusion protein;
• (f) adding a C1-C3 alcohol to the first supernatant making the second alcohol solution;
• (g) stirring the second alcohol solution forming a second stirred solution;
• (h) centrifuging the second stirred solution forming a second supernatant and a second precipitate, wherein the second precipitate comprising the target protein;
• and
• (i) separating the second precipitate comprising the target protein to yield a substantial amount of the purified target protein,
• wherein the intein is Ssp DnaB mini-intein from Synechocystis,
• wherein the volume fraction of C1-C3 alcohol added in making the first alcohol solution in step (b) is from about 50% (v/v) to about 75% (v/v), and
• wherein the volume fraction of the C1-C3 alcohol added in making the second alcohol solution in step (f) is from about 50% (v/v) to about 66.66% (v/v).