Recent advances in the production of proteins in insect and mammalian cells for structural biology.
The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.
Nettleship
Joanne E
JE
The Oxford Protein Production Facility and Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford, UK.
Assenberg
René
R
Diprose
Jonathan M
JM
Rahman-Huq
Nahid
N
Owens
Raymond J
RJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D019556:COS Cells; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014709:Vero Cells
D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins