mutagenesis using the u201c quikchange site directed mutagenesis kit u201d from stratagene ubp1mg sacii 5 u2032 ggcatagtagtattttttta ccgcgg tggtg seq id no 14 accatctaaactacattgt ubp1md sacii 5 u2032 acaatgtagtttagatggtcacca ccgcgg t seq id no 15 aaaaaaatactactatgcc using the ubp1mg and ubp1md primers marked in bold in fig1 recognition sequences for the sacii enzyme underlined were inserted into the interior of the ubp1 coding sequence thanks to this a 169 bp fragment was removed during the digestion of the pt7 7argstopubi ubp1 plasmid with the sacii restrictase this resulted in a new plasmid which we designated pt7 7argstopubi ubp1 u03b4c it contains the coding sequence shown in fig5 other plasmids coding alternate hybrid mutants according to the present invention containing the ubi sequence were produced in an analogous fashion bld21 e coli bacteria were transformed with a plasmid containing the ubp1 protease gene or one of its mutants during the culturing it was determined that the removal of the transmembrane domain facilitated the culturing and shortened the time from 30 to about 12 hours it was also observed that the cells producing the mutant according to the present invention returned to the original shape fig7 bld21 e coli bacteria containing the appropriate plasmid were cultured on lb medium containing ampicillin 50 mg ml at 25 u00b0 c over 12 h until od 600 1 and subsequently induced with the addition of iptg isopropylthiogalactoside after 2 5 h the bacteria were centrifuged the cell pellet was suspended in lysis buffer and incubated for 30 min at 20 u00b0 c triton x 100 was added to a final concentration of 1 the mixture was sonificated and centrifuged the supernatant was applied to an sp column the strong cationite sepharose.