Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters

Taylor, Troy; Denson, John-Paul; Esposito, Dominic

doi:10.1007/978-1-4939-6887-9_5

Cited by 57 publications

(50 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The BL21 STAR (DE3) E. coli strain containing rare transfer RNAs (pRare plasmid, Cm R ) was transformed with either KRAS or NF1 expression plasmids. Bacteria were cultured in Dynamite medium for protein expression ( Taylor et al, 2017 ). SPRED1 was expressed in the baculovirus expression vector system using Tni-FNL cells ( Talsania et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR

et al. 2020

Self Cite

View full text Add to dashboard Cite

SUMMARY Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

show abstract

Section: Methodsmentioning

confidence: 99%

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Proteins were expressed in E. coli as described previously (30). For insect cell expression, baculoviruses were generated by transfection of bacmid DNA into Sf9 cells, as described previously (31).…”

Section: Protein Productionmentioning

confidence: 99%

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

et al. 2019

Self Cite

View full text Add to dashboard Cite

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

show abstract

“…Given the fast growth rate (doubling time is 20 min), the low cost (the medium is inexpensive), the easiness of genetic manipulation, the well-known molecular features, the good productivity and the simple fermentation process development for manufacturing scale-up, this microorganism represents an affordable expression system for recombinant protein [43][44][45][46]. In shake flasks, E. coli generally produces a low amount of proteins (mg/L).…”

Section: Features Advantages and Disadvantages Of The E Coli Microbmentioning

confidence: 99%

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Sandomenico

Sivaccumar

Ruvo

2020

IJMS

View full text Add to dashboard Cite

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

show abstract

Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters

Cited by 57 publications

References 17 publications

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Contact Info

Product

Resources

About