Recombinant protein expression and purification of Taq DNA polymerase v1

2021 ◽  
Author(s):  
Maira Rivera ◽  
Javiera Reyes ◽  
Javiera A Avilés ◽  
Amparo Núñez ◽  
Fernan Federici ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Dna Polymerase ◽  
Recombinant Protein Expression ◽  
Recombinant Expression ◽  
Storage Conditions ◽  
Expression And Purification ◽  
Taq Dna Polymerase ◽  
Protein Expression And Purification

This is a slightly modified and simplified version of a protocol by Thomas G.W. Graham et al, which is available at https://gitlab.com/tjian-darzacq-lab/bearmix and has been described in depth in the article 10.1371/journal.pone.0246647, for the recombinant expression of a E602D mutant of Taq DNA polymerase in pET-28a that is available in Addgene (Addgene plasmid # 166944 ; http://n2t.net/addgene:166944 ; RRID:Addgene_166944). The main goal of this protocol is to eliminate the use of large volumes for dialysis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions.

Recombinant protein expression and purification of codon-optimized Pfu-Sso7d  v2

2021 ◽  
Author(s):  
Maira Rivera ◽  
Javiera Reyes ◽  
Paula Blazquez-Sanchez ◽  
Cesar A Ramirez-Sarmiento
Keyword(s):  
Pyrococcus Furiosus ◽  
Recombinant Protein Expression ◽  
Recombinant Expression ◽  
Dna Amplification ◽  
Storage Conditions ◽  
Expression And Purification ◽  
Double Stranded Dna ◽  
Protein Expression And Purification ◽  
Thermal Stability Of ◽  
Specific Sequences

This protocol has been optimized for the recombinant expression of a codon-optizimed Pfu-Sso7d DNA polymerase. This is a fusion protein composed of the Pfu enzyme from Pyrococcus furiosus for DNA amplification by PCR fused to a small 7 kDa protein from Sulfobulus solfataricus that binds to double-stranded DNA without any preference for specific sequences, thus enhancing polymerization processivity without affecting the catalytic activity or thermal stability of the enzyme. The goal of this protocol was to eliminate the use of large volumes for dyalisis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions. We also eliminated the use of DTT, which is often found in other similar protocols. The sequence plasmid encoding the codon-optimized Pfu-Sso7d enzyme used here can be found at https://benchling.com/s/seq-2TcUPjO2uMbDG5ufTQN4

scholarly journals The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e112643 ◽  
Author(s):  
Theodoros Goulas ◽  
Anna Cuppari ◽  
Raquel Garcia-Castellanos ◽  
Scott Snipas ◽  
Rudi Glockshuber ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression And Purification ◽  
System A ◽  
Protein Expression And Purification
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