Efficient production of artificially designed gelatins with a Bacillus brevis system

Artificially designed gelatins comprising tandemly repeated 30-amino-acid p eptide units derived from human alpha I collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synth esized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-m entioned strategy. Minirepeat genes could be selected in vivo from a mixtur e of every possible sequence encoding an artificial gelatin by randomly lig ating the mixed sequence unit and transforming it into Escherichia coli. La rger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins der ived from the eight-unit and six-unit repeat genes were extracellularly pro duced at the level of 0.5 g/liter and easily purified by ammonium sulfate f ractionation and anion-exchange chromatography. The purified artificial gel atins had the predicted N-terminal sequences and amino acid compositions an d a solgel property similar to that of the native gelatin. These results su ggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.