HPLC-free Protein Synthesis

HPLC-free Protein Synthesis

Author: ChemistryViews.org

The total synthesis of proteins is time consuming, requires expensive equipment, and large volumes of organic solvents. During the process, smaller protein fragments are purified by high pressure liquid chromatography (HPLC) and are added together.

Oliver Seitz and colleagues, Humboldt University of Berlin, Germany, have developed a fast and cheaper chemical total synthesis of proteins without HPLC-purification. They prepared peptide fragments by conventional solid phase synthesis and coupled them by native chemical ligation on agarose beads. The team introduced three protein tags to the ends of the protein fragments, an N-terminal His6 unit (permits selective immobilization onto agarose beds), a C-terminal peptide hydrazide (allows selective selection of ligation product), and an N-terminal 2-mercapto-2-phenyl-ethyl group (couples the two fragments). The protein tags make sure that only full length peptides and no fragments react. Incomplete protein fragments and impurities are washed out.

In three days instead of a month, the team synthesized nine 46 to 126 aa long MUC1 proteins. They were obtained in 8–33 % yield with 90–98 % purity. According to the researchers, their method simplifies and improves the throughput of protein synthesis and, therefore, has great potential for building up large protein libraries.


 

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