Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein.
Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.
Korepanova
A
A
Abbott Laboratories, Dept. R46Y, Bldg. AP10-LL8, 100 Abbott Park Road, Abbott Park, IL 60064-6098, USA.
Moore
J D
JD
Nguyen
H B
HB
Hua
Y
Y
Cross
T A
TA
Gao
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D002479:Inclusion Bodies / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine