BspK, a serine protease from the predatory bacterium Bdellovibrio bacteriovorus with utility for analysis of therapeutic antibodies
Bratanis, Eleni LU ; Molina, Henrik ; Nägeli, Andreas LU ; Collin, Mattias LU
and Lood, Rolf
LU
(2017)
In Applied and Environmental Microbiology
83(4).
- Abstract
The development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and... (More)
The development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K28 and K210 in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl2. BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K226 generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market.
(Less)
- author
- Bratanis, Eleni
LU
; Molina, Henrik
; Nägeli, Andreas
LU
; Collin, Mattias
LU
and Lood, Rolf
LU
- organization
- publishing date
- 2017
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Autoproteolysis, Bdellovibrio bacteriovorus, Biotechnology, Immunoglobulins, Serine protease, Therapeutic antibodies
- in
- Applied and Environmental Microbiology
- volume
- 83
- issue
- 4
- article number
- e03037-16
- publisher
- American Society for Microbiology
- external identifiers
-
- pmid:27940543
- wos:000393482200021
- scopus:85011966183
- ISSN
- 0099-2240
- DOI
- 10.1128/AEM.03037-16
- language
- English
- LU publication?
- yes
- id
- 6d25aee6-f14a-4d5d-8b8a-06af69e9c878
- date added to LUP
- 2017-02-23 11:14:18
- date last changed
- 2024-10-14 01:05:39
@article{6d25aee6-f14a-4d5d-8b8a-06af69e9c878,
abstract = {{<p>The development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K<sub>28</sub> and K<sub>210</sub> in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl<sub>2</sub>. BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K<sub>226</sub> generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market.</p>}},
author = {{Bratanis, Eleni and Molina, Henrik and Nägeli, Andreas and Collin, Mattias and Lood, Rolf}},
issn = {{0099-2240}},
keywords = {{Autoproteolysis; Bdellovibrio bacteriovorus; Biotechnology; Immunoglobulins; Serine protease; Therapeutic antibodies}},
language = {{eng}},
number = {{4}},
publisher = {{American Society for Microbiology}},
series = {{Applied and Environmental Microbiology}},
title = {{BspK, a serine protease from the predatory bacterium Bdellovibrio bacteriovorus with utility for analysis of therapeutic antibodies}},
url = {{http://dx.doi.org/10.1128/AEM.03037-16}},
doi = {{10.1128/AEM.03037-16}},
volume = {{83}},
year = {{2017}},
}