Modified variant bowman birk protease inhibitors
A technology of protease inhibitors and protease inhibition, which is applied in the direction of protease inhibitors, DNA/RNA fragments, endopeptidases, etc., and can solve the problems of impractical clinical application of protease inhibitors
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Embodiment 1
[0707] Production of BCE103-BBI fusion protein in Bacillus subtilis
[0708] In this example, experiments for BCE103-BBI fusion protein production in Bacillus subtilis are described. 编码用于这些实施方案中的具有C端六聚组氨酸标签的原BBI蛋白(pro-BBI)的合成基因(Operon Technologies)的DNA序列是:AACCTGCGTCTGTCTAAGCTTGGCCTGCTTATGAAATCAGACCATCAGCACAGCAATGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATAGCTGTCATAGTGCATGCAAAAGCTGTATCTGCGCCCTGAGTTATCCAGCTCAATGTTTTTGCGTCGACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGACGATAAAGAGAACCATCATCACCATCACCAT(SEQ ID NO:10)
[0709] The protein sequence of pro-BBI with a C-terminal hexahistidine tag encoded by the above synthetic gene is:
[0710] NLRLSKLGLLMKSDHQHSNDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKENHHHHHH (SEQ ID NO: 11)
[0711] The DNA sequence portion of the synthetic gene encoding the major mature form of BBI is:
[0712] GACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATGTACGAAATCAAATCCTCACACAGTGTCGGTGTTCCGATATGCGT...
Embodiment 2
[0730] Production of peptides that replace the BBI reactive site loop into BCE103-BBI fusion proteins
[0731] In this example, experiments performed to produce peptides that replace the BBI reactive site loop into BCE103-BBI fusion proteins are described. Primers are provided below, along with other sequences used in the various steps of these experiments. image 3 The 12BBIck81 sequence from the BCE103 fusion site (BamHI site) to the end of the gene is provided. The CK37281 peptide sequence (ACYNLYGWTC (SEQ ID NO: 9) was inserted into the trypsin and chymotrypsin inhibition loop.
[0732] use Site-directed mutagenesis (Stratagene), the primers used to introduce the EcoRI site in the BBI gene are:
[0733] BowBeco-F
[0734] 5'-GATATGCGTCTGAATTCCTGTCATAGTGCAT (SEQ ID NO: 17)
[0735] BowBeco-R
[0736] 5'-ATGCACTATGACAGGAATTCAGACGCATATC (SEQ ID NO: 18)
[0737] The DNA oligonucleotide sequence annealed and cloned into the BBI gene (SacI-EcoRI) to replace the trypsin...
Embodiment 3
[0978] Activation of BBI by thiol reduction / oxidant
[0979] After growth, the activity of BBI (inhibition by trypsin or chymotrypsin) is usually 5-20 times lower than that expected from the BCE103 cellulase measured in the cell-free supernatant (in the fusion protein, both molecules should be present in a 1:1 molar ratio). Increases in BBI activity in BCE103-BBI fusion proteins can be routinely obtained by adding βME, typically at a concentration of 1-4 mM, to MBD growth media approximately 14 hours after inoculation (measured by trypsin or chymotrypsin activity). inhibition). The trypsin or chymotrypsin inhibitory activity of the BBI in the fusion protein was also lower than expected when the binding peptide (eg, VegF binding peptide CK37281) replaced the trypsin or chymotrypsin reactive site loop, respectively. As in the wild-type BBI, the inhibitory activity can be increased by treatment with βME. Unexpectedly, in these experiments, other thiol reducing agents (e.g., ...
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