of physiologically active proteins substantially free of methionine at the n terminus application_number US 53961290 A description the following examples and reference examples illustrate the present invention in further detail the transformants disclosed in the examples have been deposited with the fermentation research institute fri agency of industrial science and technology ministry of international trade and industry and the institute for fermentation osaka ifo under the deposit numbers specified in table 2 table 2______________________________________ deposited with fritransformant date of deposition ifo______________________________________escherichia coli ferm bp 852 ifo 14437n4830 ptb285 apr 30 1985 escherichia coli ferm bp 628 ifo 14299dh1 ptf4 apr 6 1984 escherichia coli ferm bp 967 ifo 14422c 4 ptf4 feb 16 1985 ______________________________________ a 50 ml portion of a medium prepared by adding 50 mg liter of sodium ampicillin and 15 mg liter of tetracycline hydrochloride to l medium 10 g liter bactotryptone 5 g liter bacto yeast extract 5 g liter sodium chloride was inoculated with escherichia coli n4830 ptb285 obtained in reference example 1 ii followed by overnight incubation at 37 u00b0 c with rotation and shaking the culture broth was transferred to a 5 liter jar fermenter containing 2 5 liters of modified m 9 medium supplemented with one or more metal salts as specifically given in table 3 and cultivation was started at a rate of aeration of 2 5 liters minute a rate of stirring of 1 000 rpm and a temperature of 30 u00b0 c in the middle of cultivation when the growth reached 1 000 klett units the temperature was shifted up to 42 u00b0 c and after 4 hours of continued incubation cells were harvested and frozen for each culture broth the frozen cells.