Production of membrane proteins in Escherichia coli and Lactococcus lactis.
As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis.
Geertsma
Eric R
ER
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, University of Groningen, The Netherlands.
Poolman
Bert
B
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015854:Up-Regulation
D008565:Membrane Proteins