The E. coli single protein production system for production and structural analysis of membrane proteins

Mao, Lili; Vaiphei, S. Thangminlal; Shimazu, Tsutomu; Schneider, William M.; Tang, Yuefeng; Mani, Rajeswari; Roth, Monica J.; Montelione, Gaetano T.; Inouye, Masayori

doi:10.1007/s10969-009-9070-2

Cited by 12 publications

(5 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This system has a remarkable ability to suppress isotope dilution, and the culture can be condensed ≤150-fold, because most cell activities are arrested by the action of MazF RNase during the desired protein overexpression period . Using this system, isotopically enriched membrane protein was overproduced, and NMR signals of the target protein were measured without undesired off-target signals. , It was noted that NMR signals of the target membrane protein were detected in the crude membrane fraction of the E. coli host cells. Although the SPP method has hitherto not been used for amino acid selective 13 C labeling, our 13 C labeling method will work with the SPP system.…”

Section: Discussionmentioning

confidence: 99%

“…38 Using this system, isotopically enriched membrane protein was overproduced, and NMR signals of the target protein were measured without undesired off-target signals. 39,40 It was noted that NMR signals of the target membrane protein were detected in the crude membrane fraction of the E. coli host cells. Although the SPP method has hitherto not been used for amino acid selective 13 C labeling, our 13 C labeling method will work with the SPP system.…”

Section: ■ Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

et al. 2018

View full text Add to dashboard Cite

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. CarbonylC atoms experience less isotope scrambling than the main-chain N atoms do. However, little is known about the side-chainC atoms. Here, the C scrambling profiles of the Cα and side-chain carbons were investigated forN scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in Cα andC side-chain labeling than in N labeling. We utilized this reduced scrambling-prone character ofC as a simple and efficient method for amino acid selective C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, theC labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. H-N heteronuclear single-quantum coherence signals of the N scrambling-prone amino acid were also easily filtered usingN-{Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Although membrane proteins represent between 20-30% of all the genes in a genome (Wallin and von Heijne, 1998), relatively few structures have been solved. In fact, only around 2% of all the structures in the Protein Data Bank (PDB) are of membrane proteins (Mao et al, 2009; see also http://blanco.biomol.uci.edu/mpstruc/listAll/list and http:// pdbtm.enzim.hu/). The reasons for this scarcity of data are manifold, but can mainly be ascribed in part to the fact that the level of expression of recombinant membrane proteins (in heterologous, or homologous, systems) is significantly lower than that of cytosolic proteins and in many cases they are toxic to the host cell (Wagner et al, 2007(Wagner et al, , 2006.…”

Section: Introductionmentioning

confidence: 99%

High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Bruni

Kloss

2013

CP Protein Science

View full text Add to dashboard Cite

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable.

show abstract

“…Addition of 13 C-glucose in the medium at the time of expression can therefore allow selective isotopic enrichment of the protein of interest with 13 C without incorporation in any other cellular proteins(Mao et al 2010). The presence of a specifically isotopically-enriched protein in the context of an intact cell provides exciting opportunities in biophysical studies, particularly for NMR experiments.…”

Section: Introductionmentioning

confidence: 99%

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

et al. 2011

Self Cite

View full text Add to dashboard Cite

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

show abstract

The E. coli single protein production system for production and structural analysis of membrane proteins

Cited by 12 publications

References 11 publications

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Contact Info

Product

Resources

About

The E. coli single protein production system for production and structural analysis of membrane proteins

Cited by 12 publications

References 11 publications

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Contact Info

Product

Resources

About

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance

Amino Acid Selective ¹³C Labeling and ¹³C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance