Production of a bioactive peptide (IIAEK) in Escherichia coli using soybean proglycinin A1ab1b as a carrier.
To produce large amounts of a peptide of fewer than 10 amino acid residues, construction of a gene encoding multimers of the small peptide is necessary. For this study a method was developed to facilitate the gene construction of high multimers of a small peptide with one step of cloning. A hypocholesterolemic peptide, IIAEK, from cow's milk beta-lactoglobulin was used as a model peptide for the construction of a gene encoding multimers of IIAEK and for the production of the peptide. Two systems for direct expression of 28-mers of IIAEK sequences (28IIAEK) and expression of 34 IIAEK sequences (4 IIAEK sequences in each of the disordered regions I, II, and III and 14 and 8 IIAEK sequences in disordered regions IV and V, respectively) in a mutant of soybean proglycinin A1aB1b lacking 31 residues in disordered region IV [A1aB1b(Delta31)-34IIAEK] were used. The protein produced from both systems formed inclusion bodies. The expression level of A1aB1b(Delta31)-34IIAEK was 29.9% of the total cell proteins and that of the 28IIAEK was 2.0%. The insoluble A1aB1b(Delta31)-34IIAEK was digested by trypsin without any help from urea or chemicals, and the produced IIAEK was purified using an octadecyl silica column. The yield of IIAEK was 58.6%. The results showed that A1aB1b as a carrier of multiple peptides and use of an Escherichia coli expression system are suitable for production of bioactive peptide.
Prak
Krisna
K
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Utsumi
Shigeru
S
D000924:Anticholesteremic Agents; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D030262:Soybean Proteins / Q000235:genetics*; D014357:Trypsin / Q000378:metabolism
D000924:Anticholesteremic Agents; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D030262:Soybean Proteins; C516979:lactostatin; C405345:proglycinin A(1a)B(1b), soybean; D014357:Trypsin