Calmodulin (CaM) is a calcium-sensing protein that plays a role in regulating enzyme activity. Often, CaM binds to a disordered region and causes the region to have an ��-helical secondary structure. PEP-19 is a completely disordered 62 amino acid protein that is found in purkinje cells in the cerebellum. When PEP-19 binds to CaM, the rate at which calcium binds increases altering calcium binding kinetics. To investigate PEP-19’s conformational ensemble, we made and purified PEP-19 using <i>E.coli</i> and an expression method called autoinduction. Autoinduction is a unique method that uses specific proportions and concentrations of glycerol, glucose, and lactose, 5052, to cause the production of PEP-19 to occur without needing to monitor cell growth. Traditionally, autoinduction utilizes a specific autoinduction media which contains a complex variety of ingredients making it costly and troublesome to make. The original way we prepared PEP-19 was by using an IPTG method, which itself is inconvenient and time consuming due to the need to monitor cell growth. The IPTG method often requires the use of a simple, cost effective media called Terrific Broth (TB) that utilizes glycerol as the carbohydrate source. We hypothesized that we could utilize TB for autoinduction by replacing the standard glycerol with the 5052 mixture from autoinduction. Here we compare the expression of both CaM and PEP-19 utilizing the IPTG method, standard induction method, and autoinduction method using TB and 5052 to determine if utilization of TB and 5052 can provide a convenient, cost effective alternative to the IPTG and standard autoinduction methods.

Methods of expresion and purification of PEP-19: A calmodulin binding partner

253rd National Meeting (2017)

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