Membrane Protein Production in Lactococcus lactis for Structural Studies.

The expression and downstream purification of membrane proteins is the prerequisite for biophysical and structural studies of this major source of therapeutic targets. The gram-positive bacterium Lactococcus lactis is an attractive option for heterologous membrane protein expression and purification thanks to advantageous characteristics such as mild proteolytic activity and small genome size. Vectors designed for gene transcription under the control of inducible promoters are readily available. Specifically, the tightly regulated nisin-inducible gene expression system (NICE) allows to fine-tune the overexpression of different gene products. The expressed protein engineered with a suitable tag can be readily detected and purified from crude membrane extracts. The purpose of this protocol chapter is to detail the procedures of cloning, expression, isolation of the membrane vesicles, and affinity purification of a membrane protein of interest in L. lactis.