Recombinant expression and coexpression of oyster defensin and proline‐rich peptide in Komagataella phaffii

2021 ◽  
Author(s):  
Mine Erdem Büyükkiraz ◽  
Zülal Kesmen
Keyword(s):  
Recombinant Expression ◽  
Komagataella Phaffii ◽  
Proline Rich Peptide

scholarly journals Recombinant Co-Expression of Collagen A1 (I) Fragment with the Prolyl 4-Hydroxylases (P4H) Subunits in Komagataella phaffii

2020 ◽  
pp. 65-74
Author(s):  
Esra Avci ◽  
Zülal Kesmen
Keyword(s):  
Target Genes ◽  
Recombinant Expression ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
E Coli ◽  
Recombinant Strains ◽  
Komagataella Phaffii ◽  
Standard Quality ◽  
Collagen Fragment

Recombinant collagen and collagen-like products are increasingly replacing animal-sourced collagen that is difficult to produce in safe and standard quality. In this study to produce hydroxylated collagen, a 400 base pair collagen fragment of the bovine COL1A1 gene was co-expressed with prolyl-4-hydroxylase subunit α (P4Hα) and prolyl-4-hydroxylase subunit β(P4Hβ) encoding the P4H enzyme in Komagataella phaffii. For this purpose, each target gene was inserted into the pPICZαA vector and then cloned in E. coli DH5α cells. Subsequently, co-expression vectors were constructed using recombinant vectors isolated from positive clones according to the in vitro multimer ligation method. All recombinant expression and co-expression vectors were transformed into K. phaffii X33 cells by electroporation. The results of reverse transcriptase-polymerase chain reaction (PCR) proved that all target genes were transcribed by recombinant strains. The expression of recombinant proteins was performed for 96 hours by methanol-fed cultivation, and the concentration of the purified proteins from the culture medium was measured by the His-Tag enzyme-linked immunosorbent assay (ELISA) method. The concentrations of rP4Hα and rP4Hβ, and rCol1 proteins expressed individually by recombinant strains were determined to be 10.69 µg/L, 10.74 µg/L, and 8.61 µg/L, respectively, while the concentrations of co-expressed rP4Hα/β and rP4Hα/β/rCol1 proteins were 7.82 µg/L and 5.02 µg/L, respectively. These results showed that the target genes were successfully expressed and co-expressed in the recombinant K. phaffii cell.

Recombinant expression, purification and functional impact of CTGF/CCN2 and NOV/CCN3 on liver fibrogenesis

2011 ◽  
Vol 49 (01) ◽  
Author(s):  
W Bohr ◽  
S Lux ◽  
E Borkham-Kamphorst ◽  
E Van de Leur ◽  
M Kupper ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Functional Impact ◽  
Liver Fibrogenesis

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line

2018 ◽  
Vol 00 (00) ◽  
pp. 0-0
Author(s):  
Dongdong Li ◽  
Dongdong Li ◽  
Tai An ◽  
Xiao Liu ◽  
Bin Yin ◽  
...  
Keyword(s):  
Cell Line ◽  
Recombinant Expression ◽  
Expression And Purification

Recombinant Expression, Purification and Characterisation of the Hmg Domain of Human Sry

2003 ◽  
Vol 10 (3) ◽  
pp. 281-286 ◽  
Author(s):  
Sabine Kelly ◽  
Julia Yotis ◽  
Mary Macris ◽  
Vincent Harley
Keyword(s):  
Recombinant Expression

scholarly journals Two-step functional screen on multiple proteinaceous substrates reveals temperature-robust proteases with a broad-substrate range

2021 ◽  
Author(s):  
Antonio García-Moyano ◽  
Yuleima Diaz ◽  
José Navarro ◽  
David Almendral ◽  
Pål Puntervoll ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Skim Milk ◽  
In Silico Analysis ◽  
Prolonged Incubation ◽  
Genomic Libraries ◽  
Functional Screen ◽  
Biomass Processing ◽  
Screening Parameter ◽  
Marine Metagenome ◽  
Functional Screens

Abstract To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9–11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. Key points • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.

scholarly journals Process Simulation and Techno-Economic Analysis of Large-Scale Bioproduction of Sweet Protein Thaumatin II

Foods ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 838
Author(s):  
Kirolos D. Kelada ◽  
Daniel Tusé ◽  
Yuri Gleba ◽  
Karen A. McDonald ◽  
Somen Nandi
Keyword(s):  
Economic Analysis ◽  
Process Simulation ◽  
Large Scale ◽  
Recombinant Expression ◽  
Molecular Farming ◽  
Consumer Preference ◽  
Production Method ◽  
Long Term Effects ◽  
Adverse Health Effects ◽  
Sweet Proteins

There are currently worldwide efforts to reduce sugar intake due to the various adverse health effects linked with the overconsumption of sugars. Artificial sweeteners have been used as an alternative to nutritive sugars in numerous applications; however, their long-term effects on human health remain controversial. This led to a shift in consumer preference towards non-caloric sweeteners from natural sources. Thaumatins are a class of intensely sweet proteins found in arils of the fruits of the West-African plant Thaumatococcus daniellii. Thaumatins’ current production method through aqueous extraction from this plant and uncertainty of the harvest from tropical rainforests limits its supply while the demand is increasing. Despite successful recombinant expression of the protein in several organisms, no large-scale bioproduction facilities exist. We present preliminary process design, process simulation, and economic analysis for a large-scale (50 metric tons/year) production of a thaumatin II variant using several different molecular farming platforms.

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