scholarly journals InteBac - An integrated bacterial and baculovirus expression vector suite

2020 ◽  
Author(s):  
Veronika Altmannova ◽  
Andreas Blaha ◽  
Susanne Astrinidis ◽  
Heidi Reichle ◽  
John R. Weir
Keyword(s):  
Recombinant Protein ◽  
Insect Cell ◽  
Expression System ◽  
Integrated System ◽  
Expression Vectors ◽  
E Coli ◽  
Baculovirus Expression ◽  
Biological Studies ◽  
Cell Expression ◽  
Insect Cell Expression

The successful production of recombinant protein for biochemical, biophysical and structural biological studies critically depends on the correct expression organism. Currently the most commonly used expression organisms for structural studies are E. coli (ca. 70% of all PDB structures) and the baculovirus/ insect cell expression system (ca. 5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows the simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N- and C-terminal affinity, solublisation and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system

1990 ◽  
Vol 167 (3) ◽  
pp. 1154-1161 ◽  
Author(s):  
Miklós Sárvári ◽  
György Csikós ◽  
Miklós Sass ◽  
Péter Gál ◽  
Verne N. Schumaker ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Insect Cell ◽  
Expression System ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

Protein production using the baculovirus-insect cell expression system

2013 ◽  
Vol 30 (1) ◽  
pp. 1-18 ◽  
Author(s):  
A. Contreras-Gómez ◽  
A. Sánchez-Mirón ◽  
F. García-Camacho ◽  
E. Molina-Grima ◽  
Y. Chisti
Keyword(s):  
Insect Cell ◽  
Protein Production ◽  
Expression System ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

scholarly journals Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jan Weber ◽  
Zhaopeng Li ◽  
Ursula Rinas
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Limiting Factors ◽  
Expression Vectors ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Metabolic Burden ◽  
Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

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