High-Level Cell-Free Protein Expression from PCR-Generated DNA Templates

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

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Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽

10.1080/15476286.2020.1868149 ◽

2021 ◽

pp. 1-12

Author(s):

Yaroslav Chushak ◽

Svetlana Harbaugh ◽

Kathryn Zimlich ◽

Bryan Alfred ◽

Jorge Chávez ◽

...

Keyword(s):

Protein Expression ◽

Expression Systems ◽

Free Protein

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Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Journal of Visualized Experiments ◽

10.3791/59495 ◽

2019 ◽

Cited By ~ 3

Author(s):

Daniel J. Wiegand ◽

Henry H. Lee ◽

Nili Ostrov ◽

George M. Church

Keyword(s):

Protein Expression ◽

Free Protein

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High-level extracellular protein expression in Bacillus subtilis by optimizing strong promoters based on the transcriptome of Bacillus subtilis and Bacillus megaterium

Protein Expression and Purification ◽

10.1016/j.pep.2018.06.006 ◽

2018 ◽

Vol 151 ◽

pp. 72-77 ◽

Cited By ~ 6

Author(s):

Xin Liu ◽

Hai Wang ◽

Bin Wang ◽

Li Pan

Keyword(s):

Bacillus Subtilis ◽

Protein Expression ◽

Bacillus Megaterium ◽

Extracellular Protein ◽

High Level

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Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

BIO Web of Conferences ◽

10.1051/bioconf/20202004004 ◽

2020 ◽

Vol 20 ◽

pp. 04004

Author(s):

Ahmad Pandu Satria Wiratama ◽

Aris Haryanto

Keyword(s):

Protein Expression ◽

Newcastle Disease ◽

Recombinant Plasmid ◽

Expression System ◽

Free Protein ◽

F Protein ◽

E Coli ◽

Protein Expression System ◽

Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

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High-Level Conversion of l-lysine into Cadaverine by Escherichia coli Whole Cell Biocatalyst Expressing Hafnia alvei l-lysine Decarboxylase

Polymers ◽

10.3390/polym11071184 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1184 ◽

Cited By ~ 7

Author(s):

Kim ◽

Baritugo ◽

Oh ◽

Kang ◽

Jung ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Pyridoxal Phosphate ◽

Lysine Decarboxylase ◽

Hafnia Alvei ◽

Whole Cell ◽

E Coli ◽

Whole Cell Biocatalyst ◽

High Level ◽

Whole Cell Bioconversion

Cadaverine is a C5 diamine monomer used for the production of bio-based polyamide 510. Cadaverine is produced by the decarboxylation of l-lysine using a lysine decarboxylase (LDC). In this study, we developed recombinant Escherichia coli strains for the expression of LDC from Hafnia alvei. The resulting recombinant XBHaLDC strain was used as a whole cell biocatalyst for the high-level bioconversion of l-lysine into cadaverine without the supplementation of isopropyl β-d-1-thiogalactopyranoside (IPTG) for the induction of protein expression and pyridoxal phosphate (PLP), a key cofactor for an LDC reaction. The comparison of results from enzyme characterization of E. coli and H. alvei LDC revealed that H. alvei LDC exhibited greater bioconversion ability than E. coli LDC due to higher levels of protein expression in all cellular fractions and a higher specific activity at 37 °C (1825 U/mg protein > 1003 U/mg protein). The recombinant XBHaLDC and XBEcLDC strains were constructed for the high-level production of cadaverine. Recombinant XBHaLDC produced a 1.3-fold higher titer of cadaverine (6.1 g/L) than the XBEcLDC strain (4.8 g/L) from 10 g/L of l-lysine. Furthermore, XBHaLDC, concentrated to an optical density (OD600) of 50, efficiently produced 136 g/L of cadaverine from 200 g/L of l-lysine (97% molar yield) via an IPTG- and PLP-free whole cell bioconversion reaction. Cadaverine synthesized via a whole cell biocatalyst reaction using XBHaLDC was purified to polymer grade, and purified cadaverine was successfully used for the synthesis of polyamide 510. In conclusion, an IPTG- and PLP-free whole cell bioconversion process of l-lysine into cadaverine, using recombinant XBHaLDC, was successfully utilized for the production of bio-based polyamide 510, which has physical and thermal properties similar to polyamide 510 synthesized from chemical-grade cadaverine.

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