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 possibl e to build concatemer genes with as many as 25 repeats of the monomer unit encoding a protein-based polymer with a molecular weight of great er than 100,000 Da. This employed the use of terminal cloning adaptor oligonucleotides as chain terminators to enhance the desired polymer g ene size distribution. These genes have been expressed in Escherichia coli and the products have been purified from the culture lysates usin g a simple centrifugation method which relies upon the inverse tempera ture transitional properties of these elastomeric protein-based polyme rs. At 4 degrees C, the polymers are soluble; on raising the temperatu re above 26 degrees C, the onset temperature (T-t) for the (GVGVP)(251 ) inverse temperature transition, the polymer separates out as the mor e dense phase. Upon shifting the temperature between 4 and 37 degrees C, the recombinant elastomeric protein-based polymers undergo reversib le phase transitions from soluble (4 degrees C) to insoluble (37 degre es C) allowing their separation hom other cellular components by sever al cycles of centrifugations at alternate transitional states. Additio nal centrifugation, at a temperature just below T-t, allows for dramat ic lowering of endotoxin levels. Furthermore, many ways of varying the value of T-t, such as adding salt to lower T-t or changing the degree of ionization in polymers with functional side chains, can be used to achieve purification of more complex polymers. (C) 1996 Academic Pres s, Inc.