Two-chaperone assisted soluble expression and purification of the bacteriophage T4 long tail fibre protein gp37.
Bacteriophage T4 recognises its host cells through its long tail fibre protein gene product (gp) 37. Gp37 is a protein containing 1026 amino acids per monomer, forming a fibrous parallel homotrimer at the distal end of the long tail fibres. The other distal half-fibre protein, gp36, is much smaller, forming a trimer of 221 amino acids per monomer. Functional and structural studies of gp37 have been hampered by the inability to produce suitable amounts of it. We produced soluble gp37 by co-expression with two bacteriophage T4-encoded chaperones in a two-vector system; co-expression with each chaperone separately did not lead to good amounts of correctly folded, trimeric protein. An expression vector for the bacteriophage T4 fibrous protein chaperone gp57 was co-transformed into bacteria with a compatible bi-cistronic expression vector containing bacteriophage T4 genes 37 and 38. A six-histidine tag is encoded amino-terminal to the gp37 gene. Recombinant trimeric gp37, containing the histidine tag and residues 12-1026 of gp37, was purified from lysed bacteria by subsequent nickel-affinity, size exclusion and strong anion exchange column chromatography. Yields of approximately 4 mg of purified protein per litre of bacterial culture were achieved. Electron microscopy confirmed the protein to form fibres around 63 nm long, presumably gp36 makes up the remaining 11 nm in the intact distal half-fibre. Purified, correctly folded, gp37 will be useful for receptor-binding studies, high-resolution structural studies and for specific binding and detection of bacteria.
Bartual
Sergio Galan
SG
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela, Spain.
Garcia-Doval
Carmela
C
Alonso
Jana
J
Schoehn
Guy
G
van Raaij
Mark J
MJ
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000378:metabolism*; D008854:Microscopy, Electron; D018832:Molecular Chaperones / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D017376:Viral Tail Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D018832:Molecular Chaperones; D017376:Viral Tail Proteins