Ion Mobility Mass Spectrometry Analysis of Alternative Metal Binding Peptides for the Utilization of Cloning, Overexpression and Purification of Affinity Tagged Lactamase
Master of Science (MS)
Date of Award
Affinity tags have many applications; they can be used for tracking proteins, improving solubility, and are a tool in protein purification of recombinant proteins. Recombinant proteins have broad use as therapeutic agents for cancers, autoimmune diseases, infectious agents, viral treatments, antibodies, diabetes, and genetic disorders. Poly-histidine tag (His-tag), P (HHHHHHH), is an established affinity tag that consist of all histidine residues. This His-tag binds readily to Ni(II), Zn(II), and Co(II), and is the primary affinity tag used to track proteins during expression cloning and purification of recombinant proteins. For this research we have designed a series of alternative metal binding (amb) heptapeptides, A (HCGPYHC), C (HCGPYGC), H (DCGPYHC), I (DDGPYHC), J (DDGPYHD), K (DDGPYHD), L (HCGPFHC), N (DCGPFHC), O (HHGPYHH), Q (HHHPHHH), and R (HHGPGHH). These peptides were designed to test their binding affinity for Ni(II)-NTA, Zn(II)-NTA, and Co(II)-NTA complexes, where NTA is nitrilotriacetic acid. The six peptides that will exhibit the greatest binding affinity for the metal NTA complexes will be further tested against the control P. This testing included competitive binding studies, collision cross section, collision induced dissociation, and computational testing. The studies were tested using ion mobility - mass spectrometry from a series of samples prepared at pH 7.0-8.0 to mimic the physiological pH and purification pH using the immobilized metal affinity chromatography (IMAC). The results were analyzed by Masslynx and Driftscope. These studies are important for developing our understanding of how the peptides would compare to an established affinity tag. The results indicated that amb5 J was the most suitable peptide to act as an affinity tag and Ni(II) was the best metal to utilize in the IMAC. A J tagged lactamase recombinant enzyme underwent gene cloning, protein expression and purification. The results indicated that lactamase recombinant protein was able to be purified with further optimization of the column conditions.
Chemistry | Physical Sciences and Mathematics
Flores, Amber, "Ion Mobility Mass Spectrometry Analysis of Alternative Metal Binding Peptides for the Utilization of Cloning, Overexpression and Purification of Affinity Tagged Lactamase" (2022). Electronic Theses & Dissertations. 730.