Reference - Detail
|Author||Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J.|
|Title||Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression.|
BACKGROUND:In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.
RESULTS:Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.
CONCLUSIONS:By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.
|MeSH||Amino Acid Sequence Animals Bacterial Proteins / chemistry Base Sequence Biotechnology / methods* Cloning, Molecular Computational Biology DNA, Complementary / metabolism Escherichia coli / metabolism* Gene Expression Genetic Vectors Humans Molecular Sequence Data Oligonucleotides Protein Engineering Protein Folding Protein Structure, Tertiary Proteins / chemistry* Recombinant Fusion Proteins / chemistry|
|WOS Category||BIOTECHNOLOGY & APPLIED MICROBIOLOGY|
|DNA material||pco12 EGFR (RDB01276)|