Biotech applications of protein kinase affinity interactions
ForfatterLund, Bjarte Aarmo
Protein kinases provide one of the cell’s most important methods for signaling and control. 2% of the encoding genome consists of protein kinase genes, and from 10-50% of the proteins in the cell are phosphorylated at some point in the cell cycle. Malfunction of protein kinases is connected to several disease-conditions, most prominently cancer, Alzheimer’s and diabetes. Understanding the interactions of protein kinases gives deeper insight into the function of protein kinases, and builds a foundation for new treatments and diagnostics. A common feature of the protein kinases is their ability to bind to adenosine triphosphate (ATP). In this study a set of resin-linked ATP-analogs with distinct binding-strategies was used to probe the ATP-binding site to investigate the use as an aﬃnity-chromatography-step in the puriﬁcation of protein kinases and together with other studies of protein kinase/ligand interactions detect diﬀerences between protein kinases to give information on how to create new speciﬁc inhibitors for protein kinases. For the model-protein kinase cAMP-dependent protein kinase/protein kinase A (PKA) a speciﬁc aﬃnity-chromatography-resin with immobilized protein kinase inhibitor (PKI) was studied using diﬀerent eluants and mutants. Binding to ATP-analog-resins was observed for 70 kDa heat shock proteins (HSP70), Abelson tyrosine-protein kinase 1 (ABL1), dual speciﬁcity tyrosine-phosphorylation- regulated kinase 1A (DYRK1A) and PKA, and there were diﬀerences in which of the resins each protein bound to. Several diﬀerent mutants were tested on immobilized PKI, including one new mutant which did not show any binding to PKI. It was also shown that by using bisubstrate inhibitors as eluants, it is possible to only elute speciﬁc isoforms of PKA. The observation of distinct binding-behavior to the diﬀerently linked ATP-analogs gives indications on how new inhibitors may be designed with higher selectivity, as well as showing potential as components of a protein puriﬁcation strategy. The work on immobilized PKI with bisubstrate inhibitors as eluants revealed that it is possible to elute only the active form of PKA from the PKI-resin, simplifying the later chromatographic steps. The non-PKI-binding protein kinase A sevenfold mutant model of Aurora B (PKA Aur7)-mutant has potential use in co-expression with kinase-dead mutants to yield more stable autophosphorylated PKA it it can be shown to be active, while being easy to purify away since it does not bind to the PKI-resin as the other PKA-variants would.
Oppgaven publiseres nå etter ønske fra kandidaten. Embargodato er endret fra 2018-05-05 til 2015-11-21. 20.11.2015 MA
ForlagUniversitetet i Tromsø
University of Tromsø
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