Calmodulin (CaM) is a calcium-sensing protein that plays a role in regulating enzyme activity. Usually, CaM binds to a disordered region and causes the region to have an ��-helical secondary structure. Calcineurin (CaN) is a calcium and CaM dependent protein that activates T-cells, RAC kinase can serve as an activator for CaM dependent kinases, and PEP-19 is a disordered 62 amino acid protein that binds to CaM and alters CaM’s calcium binding kinetics. We used these four proteins to explore and develop a time and labor efficient method for producing proteins in E. coli in an undergraduate laboratory setting (research and teaching). The original method for preparing these proteins was standard IPTG induction, which is problematic for undergraduate labs due to the time-frame required for monitoring cell growth. Autoinduction is a unique method that uses specific proportions and concentrations of glycerol, glucose, and lactose, 5052, to cause the production of protein without needing to monitor cell growth. Traditionally, autoinduction utilizes a specific autoinduction media which contains a complex variety of ingredients making it costly and time consuming to make. 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 TB could be used for autoinduction by replacing the standard glycerol with the 5052 mixture from the autoinduction. Here we compare the expression of CaM, PEP-19, RAC kinase, and Calcineurin (CaN) utilizing the IPTG method, standard autoinduction 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.

Exploring efficient methods for protein production in an undergraduate lab setting

255th National Meeting (2018)

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