Expression, purification and testing of lanthanide-dependent enzymes in Methylorubrum extorquens AM1.

Abstract

With mounting evidence of the importance of lanthanide metals in biology and among diverse bacterial phyla, a platform for high-throughput microbial growth for expression and purification of lanthanide-dependent enzymes is increasingly important. Presented in this chapter is a stream-lined approach for growth of the model methylotrophic bacterium Methylorubrum extorquens AM1 for the expression of lanthanide-dependent enzymes. Growth is optimized for both high-throughput phenotypic characterization facilitating in vivo studies, as well as for scaled-up batch cultivation for enzyme purification allowing for in vitro enzymatic studies. Both approaches have been shown to be important to understanding the function and structure of these enzymes. Expression systems have been designed for production of enzymes with and without lanthanide metals, allowing for detection of lanthanide dependence. The protocol described herein is expected to accelerate the discovery of novel lanthanide-dependent enzymes and our understanding of the role of these metals in the greater biological world.