Observation of a possible pause mutant in the total synthesis of T4 lysozyme

A DNA of 495 bp coding for T4-lysozyme was chemically synthesized and cloned in Escherichia coli. On DNA sequence analysis, clones pTLY.10 and pTLY.9 were identified to contain identical and complete T4-lysozyme coding sequences except that pTLY.9 had an additional 23 bp inverted repeat DNA at the 3'-end of the coding sequence. On expression and purification under similar conditions, T4-lysozymes from these two clones showed different degrees of retention time on HPLC as well as in the rate of enzymatic reaction. We speculate that this difference could be due to the generation of a pause mutant of T4-lysozyme in pTLY.9 under the influence of 3'-inverted repeat DNA that alters the rate of protein synthesis.