Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris

Background: Coagulation factor XIIIa(FXIIIa) plays a critical role in the final stage of blood coagulation. It is extremely important in wound healing, tissue repairing and promoting cell adhesion. The deficiency of the coagulation factor can cause hemorrhage and slow wound healing. Objective: In this study, recombinant pPICZαC-FXIIIa was expressed in Pichia pastoris, purified as well as its biological activity was determined. Methods: The FXIIIa fragment obtained from the human placenta was inserted into pPICZαC to obtain pPICZαC-FXIIIa, which was transformed into X33 after linearization, and FXIIIa inserted into Pichia pastoris X33 was screened for methanol induction. The expressed product was identified by western blotting, then the supernatant was purified by affinity chromatography, and the purified product was determined by plasma coagulation experiment. Results: Polymerase Chain Reaction(PCR) showed that the FXIIIa fragment of 2250 bp was inserted successfully into pPICZαC. The expression and purification products of the same molecular weight as target protein(about 83 kDa) were obtained, which solidified significantly when reacted with plasma. Conclusion: The expression and purification products were successful, with sufficient biological activity, which can be used as a candidate FXIIIa hemostatic agent in genetic engineering.

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Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2019.1621151 ◽

2019 ◽

Vol 83 (10) ◽

pp. 1815-1821 ◽

Cited By ~ 1

Author(s):

Bangya Peng ◽

Guangpu Xue ◽

Dongfang Xu ◽

Zanjie Feng ◽

Jing Chen ◽

...

Keyword(s):

Pichia Pastoris ◽

Serine Protease ◽

Coagulation Factor ◽

Factor Xii ◽

Protease Domain ◽

Expression And Purification ◽

Serine Protease Domain

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Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0338-4 ◽

2013 ◽

Vol 171 (1) ◽

pp. 10-19 ◽

Cited By ~ 2

Author(s):

Sudheer Reddy A.R. ◽

Padikara Kutty Satheeshkumar ◽

Mookambeswaran A. Vijayalakshmi

Keyword(s):

Pichia Pastoris ◽

Factor Viii ◽

Light Chain ◽

Coagulation Factor ◽

Biologically Active ◽

Coagulation Factor Viii ◽

In Vitro Characterization

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Characterization of Mesenchymal Stem Cells Derived from Bisphosphonate-Related Osteonecrosis of the Jaw Patients’ Gingiva

Stem Cell Reviews and Reports ◽

10.1007/s12015-021-10241-8 ◽

2021 ◽

Author(s):

Mengyu Li ◽

Jiajia Wang ◽

Yejia Yu ◽

Yuqiong Zhou ◽

Yueqi Shi ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Mesenchymal Stem Cells ◽

Signaling Pathway ◽

Critical Role ◽

Osteonecrosis Of The Jaw ◽

Skin Model ◽

Tgf Β1

AbstractBisphosphonate-related osteonecrosis of the jaw (BRONJ) is a clinical condition that specifically occurs in the oral cavity, characterized by retarded wound healing in oral mucosa accelerating the exposure of bone. Moreover, the pathological mechanism remains poorly understood. Gingival mesenchymal stem cells (GMSCs) play a critical role in gingival healing and soft tissue regeneration. Although previous studies have showed that bisphosphonates (BPs) are highly toxic to healthy GMSC, there is overall lack of direct evidence demonstrating the characterization of GMSCs derived from BRONJ patients. In present study, we isolated GMSCs for the first time from the central area of BRONJ patients’ gingiva (center-BRONJ GMSCs) and the peripheral area (peri-BRONJ GMSCs), and found that they exhibited decreased proliferation, adhesion, migration capacities and underwent early apoptosis in vitro compared control GMSCs. Notably, the central and peripheral BRONJ GMSCs transplantation in a mice excisional skin model also displayed lower cell survival rate and poor healing effects than that of controls. Mechanistically, TGF-β1 signaling pathway was suppressed not only in BRONJ patients’ gingival lesions but also in BRONJ GMSCs transplantation animal model. The results above suggested that under the microenvironment of BRONJ patients, the dysfunction of GMSCs and the suppressed TGF-β1 signaling pathway may be the vital factors in impaired gingival healing, thus contributing to persistent exposure of underlying bone and development of BRONJ. This study provides new insights into the prevention for BRONJ by improving the functions of GMSCs and upregulating TGF-β1 in accelerating gingival wound healing. Graphical Abstract Schematic illustration of the dysfunction of BRONJ GMSCs in vitro and BRONJ GMSCs transplantation in a mice skin model delaying cutaneous wound healing mainly via suppressing TGF-β1 signaling pathway.

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Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris

Biologia ◽

10.2478/s11756-010-0017-7 ◽

2010 ◽

Vol 65 (2) ◽

Cited By ~ 1

Author(s):

Jiayun Qiao ◽

Bo Zhang ◽

Yiqun Chen ◽

Yunhe Cao

Keyword(s):

Bacillus Subtilis ◽

Pichia Pastoris ◽

Codon Bias ◽

De Novo ◽

Alcohol Oxidase ◽

Methanol Induction ◽

Chemical Reagents ◽

Prospective Application ◽

High Level

Abstractβ-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40°C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant β-1,3-1,4-glucanase has a prospective application in feed and brewing industries.

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