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Safar Farajnia
Biotechnology Research center, tabrizUniversity of medical Sciences
Corresponding Author
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Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.
Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations.
Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.
Keywords
Ocriplasmin, Pichia pastoris, Site-directed mutagenesis, Proteolytic activity, Autolytic activity
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Biological Procedures Online.
No community comments so far
Editorial decision: Accept
On 30 Nov, 2020
Reviewer #1 agreed
On 23 Nov, 2020
Review #1 received
Received 23 Nov, 2020
Reviewers invited
Invitations sent on 22 Nov, 2020
Editor assigned
On 18 Nov, 2020
Submission checks complete
On 18 Nov, 2020
Editor invited
On 18 Nov, 2020
Version 1
Posted 25 Aug, 2020
No comments provided
Review #3 received
Received 26 Oct, 2020
Editorial decision: Major revision
On 26 Oct, 2020
Review #1 received
Received 20 Oct, 2020
Review #2 received
Received 15 Oct, 2020
Reviewer #3 agreed
On 28 Sep, 2020
Reviewer #2 agreed
On 26 Sep, 2020
Reviewer #1 agreed
On 21 Sep, 2020
Reviewers invited
Invitations sent on 20 Aug, 2020
First submitted
On 18 Aug, 2020
Editor assigned
On 18 Aug, 2020
Submission checks complete
On 17 Aug, 2020
Editor invited
On 17 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
Learn more about our company.
A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Biological Procedures Online.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Safar Farajnia
Biotechnology Research center, tabrizUniversity of medical Sciences
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Biological Procedures Online.
No community comments so far
Editorial decision: Accept
On 30 Nov, 2020
Reviewer #1 agreed
On 23 Nov, 2020
Review #1 received
Received 23 Nov, 2020
Reviewers invited
Invitations sent on 22 Nov, 2020
Editor assigned
On 18 Nov, 2020
Submission checks complete
On 18 Nov, 2020
Editor invited
On 18 Nov, 2020
Version 1
Posted 25 Aug, 2020
No comments provided
Review #3 received
Received 26 Oct, 2020
Editorial decision: Major revision
On 26 Oct, 2020
Review #1 received
Received 20 Oct, 2020
Review #2 received
Received 15 Oct, 2020
Reviewer #3 agreed
On 28 Sep, 2020
Reviewer #2 agreed
On 26 Sep, 2020
Reviewer #1 agreed
On 21 Sep, 2020
Reviewers invited
Invitations sent on 20 Aug, 2020
First submitted
On 18 Aug, 2020
Editor assigned
On 18 Aug, 2020
Submission checks complete
On 17 Aug, 2020
Editor invited
On 17 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Biological Procedures Online.
Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.
Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations.
Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
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