title: Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline.
abstract: Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies.
authors: Seattle Structural Genomics Center for Infectious Disease (SSGCID), University of Washington, WA 98195, USA.
authors: Stephen N
authors: SN
authors: Hewitt
authors: Ryan
authors: R
authors: Choi
authors: Angela
authors: A
authors: Kelley
authors: Gregory J
authors: GJ
authors: Crowther
authors: Alberto J
authors: AJ
authors: Napuli
authors: Wesley C
authors: WC
authors: Van Voorhis
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins