Cloning, expression and purification of human PDGF-BB gene in Escherichia coli: New approach in PDGF-BB protein production

Gene Reports ◽  
2020 ◽  
Vol 19 ◽  
pp. 100653
Author(s):  
Younes Ghasemi ◽  
Mohammad Bagher Ghoshoon ◽  
Mohammad Taheri ◽  
Manica Negahdaripour ◽  
Fatemeh Nouri
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Expression And Purification ◽  
New Approach

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

scholarly journals The 2.2Å Resolution Structure of RpiB/AlsB from Escherichia coli Illustrates a New Approach to the Ribose-5-phosphate Isomerase Reaction

2003 ◽  
Vol 332 (5) ◽  
pp. 1083-1094 ◽  
Author(s):  
Rong-Guang Zhang ◽  
C.Evalena Andersson ◽  
Tatiana Skarina ◽  
Elena Evdokimova ◽  
Aled M. Edwards ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
New Approach ◽  
Resolution Structure

Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli

2014 ◽  
Vol 173 (2) ◽  
pp. 421-432 ◽  
Author(s):  
Yunsheng Yuan ◽  
Xiyuan Zhang ◽  
Shunyan Weng ◽  
Wen Guan ◽  
Di Xiang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Purity ◽  
Expression And Purification ◽  
Recombinant Mouse

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production

2016 ◽  
Vol 6 (2) ◽  
pp. 284-300 ◽  
Author(s):  
Dimitra Gialama ◽  
Kalliopi Kostelidou ◽  
Myrsini Michou ◽  
Dafni Chrysanthi Delivoria ◽  
Fragiskos N. Kolisis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Protein Production ◽  
High Level

scholarly journals Local genic base composition impacts protein production and cellular fitness

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4286 ◽  
Author(s):  
Erik M. Quandt ◽  
Charles C. Traverse ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Base Composition ◽  
Protein Production ◽  
Purifying Selection ◽  
Specific Protein ◽  
Compositional Variation ◽  
Terminal Portion ◽  
A Genome ◽  
Selection For ◽  
Protein Feature

The maintenance of a G + C content that is higher than the mutational input to a genome provides support for the view that selection serves to increase G + C contents in bacteria. Recent experimental evidence fromEscherichia colidemonstrated that selection for increasing G + C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G + C content acts across all sites within a gene or is confined to particular genic regions or nucleotide positions. We systematically altered the G + C contents of the GFP gene and assayed its effects on the fitness of strains harboring each variant. Fitness differences were attributable to the base compositional variation in the terminal portion of the gene, suggesting a connection to the folding of a specific protein feature. Variants containing sequence features that are thought to result in rapid translation, such as low G + C content and high levels of codon adaptation, displayed highly reduced growth rates. Taken together, our results show that purifying selection acting against A and T mutations most likely results from their tendency to increase the rate of translation, which can perturb the dynamics of protein folding.

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