PROTEIN SEQUENCE DETERMINATION
CONTENTS EXPRESSION SCREENING OF INTEGRAL MEMBRANE PROTEINS BY FUSION TO FLUORESCENT REPORTERS DETERGENTS IN MEMBRANE PROTEIN PURI?CATION AND CRYSTALLISATION NMR OF MEMBRANE PROTEINS: BEYOND CRYSTALS CHARACTERISATION OF CONFORMATIONAL AND LIGAND BINDING PROPERTIES OF MEMBRANE PROTEINS USING SYNCHROTRON RADIATION CIRCULAR DICHROISM (SRCD) MEMBRANE PROTEIN CRYSTALLISATION: CURRENT TRENDS AND FUTURE PERSPECTIVES CRYSTAL DEHYDRATION IN MEMBRANE PROTEIN CRYSTALLOGRAPHY NONLINEAR OPTICAL CHARACTERIZATION OF MEMBRANE PROTEIN MICROCRYSTALS AND NANOCRYSTALS EXPLOITING MICROBEAMS FOR MEMBRANE PROTEIN STRUCTURE DETERMINATION APPLICATIONS OF THE BLEND SOFTWARE TO CRYSTALLOGRAPHIC DATA FROM MEMBRANE PROTEINS SERIAL MILLISECOND CRYSTALLOGRAPHY OF MEMBRANE PROTEINS SERIAL FEMTOSECOND CRYSTALLOGRAPHY OF MEMBRANE PROTEINS BEYOND MEMBRANE PROTEIN STRUCTURE: DRUG DISCOVERY, DYNAMICS AND DIF?CULTIES EXPRESSION SCREENING OF INTEGRAL MEMBRANE PROTEINS BY FUSION TO FLUORESCENT REPORTERS ABSTRACT THE PRODUCTION OF RECOMBINANT INTEGRAL MEMBRANE PROTEINS FOR STRUCTURAL AND FUNCTIONAL STUDIES REMAINS TECHNICALLY CHALLENGING DUE TO THEIR RELATIVELY LOW LEVELS OF EXPRESSION TO ADDRESS THIS PROBLEM, SCREENING STRATEGIES HAVE BEEN DEVELOPED TO IDENTIFY THE OPTIMAL MEMBRANE SEQUENCE AND EXPRESSION HOST FOR PROTEIN PRODUCTION A COMMON APPROACH IS TO GENETICALLY FUSE THE MEMBRANE PROTEIN TO A ?UORESCENT REPORTER, TYPICALLY GREEN FLUORESCENT PROTEIN (GFP) ENABLING EXPRESSION LEVELS, LOCALIZATION AND DETERGENT SOLUBILISATION TO BE ASSESSED INITIALLY DEVELOPED FOR SCREENING THE HETEROLOGOUS EXPRESSION OF BACTERIAL MEMBRANE PROTEINS IN ESCHERICHIA COLI, THE METHOD HAS BEEN EXTENDED TO EUKARYOTIC HOSTS, INCLUDING INSECT AND MAMMALIAN CELLS OVERALL, GFP-BASED EXPRESSION SCREENING HAS MADE A MAJOR IMPACT ON THE NUMBER OF MEMBRANE PROTEIN STRUCTURES THAT HAVE BEEN DETERMINED IN THE LAST FEW YEARS KEYWORDS INTEGRAL MEMBRANE PROTEIN GREEN ?UORESCENT PROTEIN INSECT CELLS ESCHERICHIA COLI SACCHAROMYCES CEREVISIAE PICHIA PASTORIS HEK INTRODUCTION THE PRODUCTION OF RECOMBINANT INTEGRAL MEM- BRANE PROTEINS (IMPS) FOR STRUCTURAL AND FUNC- TIONAL STUDIES IS TECHNICAL CHALLENGING DUE TO LOW LEVELS OF EXPRESSION OFTEN LIMITED BY TOXICITY TO THE EXPRESSION HOST CELLS TO OVERCOME THESE LIMITATIONS SCREENING OF SEQUENCE VARIANTS EITHER ENGINEERED OR EXPLOITING THE NATURAL SEQUENCE DIVERSITY OF ORTHOLOGUES, HAS BEEN SUCCESSFULLY USED TO IMPROVE THE PRODUCTION OF MANY MEM- , GFP GFP BACTERIA ESCHERICHIA COLI IS THE MOST COMMONLY USED PROKARYOTIC HOST FOR OVEREXPRESSION OF IMPS, FOL- LOWED BY THE GRAM POSITIVE BACTERIUM, LACTOCOC- CUS LACTIS (KUNJI ET AL 2003; DREW ET AL 2006; GORDON ET AL 2008; FRELET-BARRAND ET AL 2010; CHEN 2012; KING ET AL 2015) BACTERIAL HOSTS HAVE OBVIOUS ADVANTAGES FOR THE OVER-EXPRESSION OF RECOMBINANT PROTEINS WITH RAPID GROWTH RATES, INEXPENSIVE GROWTH MEDIA AND THE EASE OF GENETIC MANIPULATION MOREOVER, THE BIOLOGY OF TRANSCRIP- TION, TRANSLATION AND INSERTION INTO MEMBRANES ARE ALSO WELL CHARACTERISED, ALLOWING MANIPULATION OF THE HOST CELL TO FACILITATE HETEROLOGOUS EXPRESSION OF PROTEINS NEVERTHELESS, THE EXPRESSION OF MEM- BRANE PROTEINS IN BACTERIA CAN BE PROBLEMATICAL FOR A NUMBER OF REASONS THE EXPRESSED PROTEIN MAY PROVE TO BE TOXIC TO THE HOST CELL (KUNJI ET AL 2003) OR SATURATE THE MEMBRANE INSERTION MACHINERY (LOLL 2003; WAGNER ET AL 2006) RARE CODONS IN THE PROTEIN OR INSUF?CIENT AMINO ACID AVAILABILITY (ANGOV ET AL 2008; MARREDDY ET AL 2010; BILL ET AL 2011) OR INSUF?CIENT MEMBRANE CAPACITY (ARECHAGA ET AL 2000) MAY ALL LIMIT THE EXPRESSION OF MEMBRANE PROTEINS IN BACTERIA THEREFORE, SCREENING FOR CORRECTLY FOLDED PROTEIN