Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified by ammonium sulfate precipitation, gel filtration, and anion and cation exchange chromatography and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Their molecular weights were determined by electrospray mass spectrometry as 6916, 6807, 7676, 7944, 7848, and 7844 D, respectively, and the sequences of the first 20 N-terminal amino acid residues of these six inhibitors were found to be identical. The complete amino acid sequence of PSTI IVa was determined. This protein comprises a total of 72 residues and has 14 cysteines, all involved in disulfide bridges. Comparison of the sequence of PSTI IVa with those of other leguminous Bowman-Birk type inhibitors revealed that PSTI could be classified as a group III inhibitor, closely related toVicia faba andVicia angustifolia inhibitors.     

Plasmid-encoded protein attenuates Escherichia coli swimming velocity and cell growth,not reprogrammed regulatory functions

In addition to engineering new pathways for synthesis, synthetic biologists rewire cells to carry out “programmable” functions, an example being the creation of wound-healing probiotics. Engineering regulatory circuits and synthetic machinery, however, can be deleterious to cell function, particularly if the “metabolic burden” is significant. Here, a synthetic regulatory circuit previously constructed to direct Escherichia coli to swim toward hydrogen peroxide, a signal of wound generation, was shown to work even with coexpression of antibiotic resistance genes and genes associated with lactose utilization. We found, however, that cotransformation with a second vector constitutively expressing GFP (as a marker) and additionally conferring resistance to kanamycin and tetracycline resulted in slower velocity (Δ~6 μm/s) and dramatically reduced growth rate (Δ > 50%). The additional vector did not, however, alter the run-and-tumble ratio or directional characteristics of H₂O₂–dependent motility. The main impact of this additional burden was limited to slowing cell velocity and growth, suggesting that reprogrammed cell motility by minimally altering native regulatory circuits can be maintained even when extraneous burden is placed on the host cell. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2778, 2019.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.