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Tytuł pozycji:

Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications.

Tytuł:
Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications.
Autorzy:
Pemberton PA; SerPlus Technology LLC, 111 Industrial Way, Suite 9, Belmont, CA, 94002, USA. Electronic address: .
Źródło:
Protein expression and purification [Protein Expr Purif] 2020 Jun; Vol. 170, pp. 105595. Date of Electronic Publication: 2020 Feb 07.
Typ publikacji:
Journal Article
Język:
English
Imprint Name(s):
Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-
MeSH Terms:
Chymotrypsin/*metabolism
Cysteine/*metabolism
Disulfides/*metabolism
Pancreatic Elastase/*metabolism
Saccharomyces cerevisiae/*genetics
Serpins/*genetics
Cell Proliferation ; Chymotrypsin/antagonists & inhibitors ; Chymotrypsin/genetics ; Cloning, Molecular ; Disulfides/chemistry ; Enzyme Assays ; Gene Expression ; Genetic Vectors/chemistry ; Genetic Vectors/metabolism ; Molecular Weight ; Oxidation-Reduction ; Pancreatic Elastase/antagonists & inhibitors ; Pancreatic Elastase/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Saccharomyces cerevisiae/enzymology ; Serpins/isolation & purification ; Serpins/metabolism
Contributed Indexing:
Keywords: Cysteine-344; Dimer; Elastase; Monomer; Oxidative inactivation; Recombinant human serpin B1
Substance Nomenclature:
0 (Disulfides)
0 (Recombinant Proteins)
0 (Serpins)
147416-07-7 (SERPINB1 protein, human)
EC 3.4.21.1 (Chymotrypsin)
EC 3.4.21.36 (Pancreatic Elastase)
K848JZ4886 (Cysteine)
Entry Date(s):
Date Created: 20200212 Date Completed: 20210107 Latest Revision: 20210107
Update Code:
20240105
DOI:
10.1016/j.pep.2020.105595
PMID:
32044416
Czasopismo naukowe
Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.
Competing Interests: Declaration of competing interest Philip A. Pemberton is affiliated with and has ownership interests in Serplus Technology LLC.
(Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

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