Abstract
We have developed an expression/mutagenesis system and a series of screening procedures for the study of structure-function relationships in human interferon-gamma (HuIFN-gamma). Here we report a preliminary evaluation of the C-terminal portion of the molecule. An expression vector, p652trp gamma, was constructed which includes (i) the HuIFN-gamma gene under control of the trp promoter, (ii) elements controlling replication of both single- and double-stranded versions of the vector DNA; and (iii) the ampicillin resistance gene. (Other vectors using these same elements were constructed but proved to be unsatisfactory, being characterized by a rapid decline, as cells containing them were passaged, in their potential to achieve high expression levels.) A mutagenesis cassette was constructed by introduction of unique restriction sites flanking the nucleotides encoding the C-terminal 23 amino acids, and this cassette was replaced with chemically synthesized, degenerate oligonucleotides by ligation. Colonies from cells transformed with the reconstructed vector were stored in LB glycerol in microtiter plates, and these were screened by hybridization with synthetic oligonucleotides. Plates were grown in minimal medium to express the en...Continue Reading
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