high yields of recombinant protein may be dependent on the growth conditions the rate of expression and the length of time used to express the at rich gene in general at rich genes appear to be expressed at a higher level in e coli during a post exponential or slowed phase of growth high level production of the encoded protein requires moderate levels of expression over an extended period e g 20 24 h of post exponential growth rather than the typical approach of high level expression during exponential growth for shorter periods e g 4 6 h in this regard it is more efficient to maintain plasmids carrying the gene of interest by maintaining constant selective pressure for the gene or its expression vector during the extended period of growth one aspect of the present invention is using an antibiotic that is not inactivated or degraded during growth of the expression host cell as is found with ampicillin this embodiment involves the expression of genes encoding resistance to kanamycin as the selective phenotype for maintaining the expression vector which comprises such kanamycin resistance genetic sequences expression of large at rich clostridial genes in e coli at levels gt 100 mg liter provided for by methods of the present invention was hitherto unknown terms as used herein are based upon their art recognized meaning and should be clearly understood by the ordinary skilled artisan raru is a recombinant protein containing the repeating units of clostridium difficile toxin a as defined by dove et al dove et al infect immun 58 480 488 1990 the nucleotide sequence encoding raru and the amino acid sequence of raru are shown in fig2 and 3 respectively the raru expressed by prsetb aru km.