br Materials and methods br Chemical synthesis br

Materials and methods
Chemical synthesis
Declaration of interest
Acknowledgements This research was supported by a grant from the National Institute for General Medicine to K.W.M. (GM 58448) and by the Department of Anesthesia, Critical Care & Pain Medicine at Massachusetts General Hospital. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. We thank Dr. Bernard Santarsiero for determination of x-ray structures of HATU synthesis 22. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817).
Introduction Integral membrane proteins (IMPs) play crucial roles in every aspect of human physiology, accounting for approximately one quarter of all open reading frames and more than half of current pharmaceutical drug targets [1,2]. However, with IMPs representing less than 1% of protein data bank entries and only 10% of those being from vertebrate origin [3,4], our structural understanding of them is lacking. This shortcoming has been the focus of a number of recent review articles which aim to identify and address the inherent biochemical properties of IMPs which make them difficult to study [[4], [5], [6], [7], [8]]. One of the fundamental problems encountered in structural studies of IMPs is simply obtaining sufficient raw material to facilitate their study. In contrast to soluble proteins which can occupy the bulk intracellular space or be secreted out of the cell, IMPs can only exist in the small volume of the lipid bilayer. In addition, due to their complex structure a number of chaperones and enzymes are required to facilitate folding, post-translational modification and transport, which creates a bottleneck when attempting to overexpress them recombinantly [4,9]. The species-specific nature of this cellular machinery and lipid bilayer composition also precludes the use of economical and productive prokaryotic expression platforms. While most studies still rely on lower eukaryotes such as yeast and insect cells, it is generally accepted that faithful replication of in vivo structure and function can only be assured when higher eukaryotes are used [10]. Despite their relatively slow growth and high cost of culturing there has been a growing trend in the use of Human Embryonic Kidney 293 (HEK293) cells when performing structural studies on mammalian IMPs [7]. Great advancements have been made in the recombinant expression of IMPs in recent years, as evidenced by the number of solved mammalian membrane protein crystal structures from HEK293 sourced protein [11]. Most of these studies have relied on tetracycline inducible stable clones which are unmatched in their ability to produce large quantities of high quality protein [[12], [13], [14], [15], [16]], having also found use in other avenues with high protein requirements such as photoaffinity labelling [[17], [18], [19]]. A major limitation to the progress of such studies however is the time and cost involved in the production of stable clones. Upon transfection and the generation of clonal populations, dozens of candidates must be screened by polymerase chain reaction (PCR) and western blot (WB), a process which routinely takes several months to complete [20]. Many studies have subsequently turned to transient transfections as the source of protein for structural studies, predominantly via baculovirus infection of mammalian cells (BacMam) [[21], [22], [23], [24], [25]]. Even this technique is limited by its complexity however, requiring an additional step of viral particle amplification and harvesting from Sf9 insect cells prior to transfection of HEK293. Finally, direct transfection with chemical reagents, although lower yielding, has facilitated the successful solving of several IMP crystal structures in the past two years [[26], [27], [28], [29]]. This technique is also routinely used for small scale screening of modified proteins to identify candidates with adequate conformity and stability prior to attempting crystallisation [7,[30], [31], [32], [33]].