Solid Phase Peptide Synthesis: The Functionality of Heptapeptides with Metal Ion Binding Properties in Protein Purification
Poster Number
8
Format
Poster Presentation
Faculty Mentor Name
Jianhua Ren
Faculty Mentor Department
Chemistry
Graduate Student Mentor Name
Yuntao Zhang
Graduate Student Mentor Department
Chemistry
Abstract/Artist Statement
Introduction:
Proteins contain amino acids that facilitate biological processes; therefore, most diseases are due to protein dysfunction. To understand protein dysfunction, the synthesis and purification of recombinant proteins is essential for biomedical research, biotherapeutics and bioengineering. A target protein containing a side chain of histidine residues is purified by affinity chromatography that causes the His-tagged protein to be isolated. In order to enhance the efficiency and specificity of protein purification by using columns, the peptide ac-Cys1-Cys2-Gly3-Pro4-Tyr5-Cys6-Cys7-NH2 is selected to bind to the column. The peptide is synthesized by solid phase peptide synthesis (SPPS). The histidine tags allow the peptide to bind to specific resins loaded with metal ions: Zn2+ and Ni2+. Metal ions bind tightly to functional groups of histidine side chains, thus possessing high affinity for the His-tagged protein and serving as a stationary phase within the column that results in protein purification.
Methods:
In SPPS, rink amide resin is used as structural support to form peptide bonds between amino acids from the C to N terminus. Deprotection removes the fluorenylmethyloxycarbonyl (Fmoc) from the N-terminus of an amino acid to allow subsequent amino acids to attach. Next, washing using methanol, dichloromethane, and dimethylformaide (DMF) removes unreacted reagents. In coupling, diisopropylethylamine, hexafluorophosphate benzotriazole tetramethyl uronium, and DMF activate the new amino acid in preparation for attachment. The first three steps are repeated until the desired peptide is synthesized. Lastly, cleavage removes the Fmoc protecting groups and the peptide from the solid support, followed by acetylation that replaces the N terminus amine with an acetyl group. The product’s purity is analyzed by mass spectrometry.
Results:
The peptide ac-Cys1-Cys2-Gly3-Pro4-Tyr5-Cys6-Cys7-NH2 was successfully synthesized in the laboratory. Further analysis of the peptide is being performed through molecular modeling that will reveal the conformation of the peptide upon binding to metal ions: Zn2+ and Ni2+.
Location
University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211
Start Date
24-4-2021 1:00 PM
End Date
24-4-2021 2:15 PM
Solid Phase Peptide Synthesis: The Functionality of Heptapeptides with Metal Ion Binding Properties in Protein Purification
University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211
Introduction:
Proteins contain amino acids that facilitate biological processes; therefore, most diseases are due to protein dysfunction. To understand protein dysfunction, the synthesis and purification of recombinant proteins is essential for biomedical research, biotherapeutics and bioengineering. A target protein containing a side chain of histidine residues is purified by affinity chromatography that causes the His-tagged protein to be isolated. In order to enhance the efficiency and specificity of protein purification by using columns, the peptide ac-Cys1-Cys2-Gly3-Pro4-Tyr5-Cys6-Cys7-NH2 is selected to bind to the column. The peptide is synthesized by solid phase peptide synthesis (SPPS). The histidine tags allow the peptide to bind to specific resins loaded with metal ions: Zn2+ and Ni2+. Metal ions bind tightly to functional groups of histidine side chains, thus possessing high affinity for the His-tagged protein and serving as a stationary phase within the column that results in protein purification.
Methods:
In SPPS, rink amide resin is used as structural support to form peptide bonds between amino acids from the C to N terminus. Deprotection removes the fluorenylmethyloxycarbonyl (Fmoc) from the N-terminus of an amino acid to allow subsequent amino acids to attach. Next, washing using methanol, dichloromethane, and dimethylformaide (DMF) removes unreacted reagents. In coupling, diisopropylethylamine, hexafluorophosphate benzotriazole tetramethyl uronium, and DMF activate the new amino acid in preparation for attachment. The first three steps are repeated until the desired peptide is synthesized. Lastly, cleavage removes the Fmoc protecting groups and the peptide from the solid support, followed by acetylation that replaces the N terminus amine with an acetyl group. The product’s purity is analyzed by mass spectrometry.
Results:
The peptide ac-Cys1-Cys2-Gly3-Pro4-Tyr5-Cys6-Cys7-NH2 was successfully synthesized in the laboratory. Further analysis of the peptide is being performed through molecular modeling that will reveal the conformation of the peptide upon binding to metal ions: Zn2+ and Ni2+.