Title

Solid Phase Peptide Synthesis: The Functionality of Heptapeptides with Metal Ion Binding Properties in Protein Purification

Poster Number

8

Lead Author Major

Biochemistry

Lead Author Status

Senior

Second Author Major

Pre-pharmacy

Second Author Status

Sophomore

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

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Apr 24th, 1:00 PM Apr 24th, 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+.