The nucleotide sequence of gene 21 of bacteriophage T4 coding for the prohead protease

We have sequenced gene 21 coding for the bacteriophage T4 prohead protease. The sequence codes for a protein of 212 amino acids (aa) with an Mr of 23,251. A second possible in-frame initiation site was also found which would code for an Mr 18,440 protein. Evidence is presented that this second site is used in vivo. The only striking homology of gp21 to other proteins is with the serine proteases. The protein is homologous to a short aa sequence around the active site, but has a His where the active site Ser is normally found. However, mutation of this His to Ser gave a functional protein that could not be inhibited by serine protease inhibitors. We have located three sites in the gene that give rise to temperature-sensitive mutations. One of these is towards the N-terminus of the gene, the other two flank the region that shows homology with serine proteases. Attempts to overproduce the protein in Escherichia coli failed due to the extreme lability of the enzyme. A frame-shift mutation in the gene was therefore constructed which allowed the synthesis of large amounts of a stable N-terminal fragment of the protein.