MUTANTS OF HUMAN TUMOR-NECROSIS-FACTOR - PREPARATION AND PROPERTIES

Using polymerase chain reaction, a number of mutant genes encoding hum an tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained. The mutant proteins (muteins) contained poi nt mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H -F144L; and deletion of four amino acid residues 67-70. The mutant gen es were expressed in E. coli under the control of constitutive promote rs. A simple purification method for the muteins was developed and the ir physicochemical properties were studied. All the muteins obtained, except F144L and I118A, were shown by CD and cross-linking to form a s patial structure similar to that of the native TNF-alpha. The collecti on of muteins was characterized by their biological activity. Mutants R32H and A33S exerted a decreased cytotoxicity against murine fibrobla st cell line L929, whereas point mutant F144L and double mutant R32H-F 144L were essentially inactive.