Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP.
By constructing a basic gene unit encoding (GVGVP)10, it was possible to build concatemer genes with as many as 25 repeats of the monomer unit encoding a protein-based polymer with a molecular weight of greater than 100,000 Da. This employed the use of terminal cloning adaptor oligonucleotides as chain terminators to enhance the desired polymer gene size distribution. These genes have been expressed in Escherichia coli and the products have been purified from the culture lysates using a simple centrifugation method which relies upon the inverse temperature transitional properties of these elastomeric protein-based polymers. At 4 degrees C, the polymers are soluble; on raising the temperature above 26 degrees C, the onset temperature (Tt) for the (GVGVP)251 inverse temperature transition, the polymer separates out as the more dense phase. Upon shifting the temperature between 4 and 37 degrees C, the recombinant elastomeric protein-based polymers undergo reversible phase transitions from soluble (4 degrees C) to insoluble (37 degrees C) allowing their separation from other cellular components by several cycles of centrifugations at alternate transitional states. Additional centrifugation, at a temperature just below Tt, allows for dramatic lowering of endotoxin levels. Furthermore, many ways of varying the value of Tt, such as adding salt to lower Tt or changing the degree of ionization in polymers with functional side chains, can be used to achieve purification of more complex polymers.
McPherson
D T
DT
Laboratory of Molecular Biophysics, University of Alabama at Birmingham, 35294-0019, USA.
Xu
J
J
Urry
D W
DW
D000595:Amino Acid Sequence; D003300:Copper / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D012995:Solubility; D013696:Temperature
D004731:Endotoxins; D009842:Oligopeptides; D011994:Recombinant Proteins; C102077:glycyl-valyl-glycyl-valyl-proline; D003300:Copper