Efficient production of artificially designed gelatins with a Bacillus brevis system.
Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest 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.
Kajino
T
T
Toyota Central Research and Development Laboratories, Inc., Nagakute, Aichi 480-1192, Japan. e0846@mosk.tytlabs.co.jp
Takahashi
H
H
Hirai
M
M
Yamada
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005780:Gelatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014169:Transformation, Bacterial
D005780:Gelatin