SOLID- LIQUID SEPARATION OF CSPB50TEV-TERIPARATIDE

 6 amino acids in N-terminal of CspB solubilise above pH7 and insolubilize below pH6.
 The CspB50 tag, consisting of 50 amino acid residues of the N-terminal CspB, is a versatile
pH-responsive tag for the precipitation-redissolution of Teriparatide and Bivalirudin, as well
as proinsulin etc.
 purification by solid-liquid separation of the fused proteins from a cell lysate of E. coli, and a culture supernatant of C.
glutamicum, yeast or Chinese hamster ovary (CHO) cells
 the impurities have a strong absorption and emit fluorescence, which hampers the further analysis of target proteins by
spectroscopic methods.
 Use of additives is a good way to overcome aggregation and dispersion of protein and
solutes
 Arg (Arginine) is one of the best suited examples for the purification.
 The main mechanism behind: i) guanidium of Arg interact with insoluble region of the solute
through a π – cation interaction. ii) Charged α- amino atom interacts electrostatically with
guanidium group.
 The culture broth of the cultured cells expressing tagged proteins is collected and filtered
and poured into culture tubes.
 Arginine additive was added to the tubes and pH adjusted to 4.5 ˗˗˃ Centrifuged and
supernatant was removed ˗˗˃ The precipitate was redissolved in PBS (pH 8.0)
 Before adjusting pH 4.5, the culture colour was brown due to presence of metabolites, like
carotenoids C50. After pH adjustment, precipitation is successful, and precipitate is of brown
colour.
 With additive (Arg), in the dissolution step, the impurities get dissolved.
