High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system

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Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in E. coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible.Here we use a quantitative, high throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove 4 of the 5 arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 °C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of ~400 mg/ L of expression culture (~15 mg pure TEV protease per g of E. coli cell paste). Methods for producing glutathione S-transferase tagged TEV with similar yields (~12 mg pure protease fusion per g of E. coli cell paste) are also reported.

Section: Tev Protease Expression Vectorsmentioning

confidence: 99%

Section: Tev Protease Expression Vectorsmentioning

confidence: 99%

Section: Tev Protease Expression Vectorsmentioning

confidence: 99%

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A combined approach to improving large-scale production of tobacco etch virus protease

Blommel

Fox

2007

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258

“…The vectors use the viral T5 promoter under control of an engineered double lac operator for recombinant gene expression [24]. The plasmid backbones also provide the strong lacI q promoter for elevated expression of the lac repressor, LacI.…”

Section: Expression Vectorsmentioning

confidence: 99%

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Sobrado

Goren

James

et al. 2008

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A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of ~18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per]of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

“…Thus the yield of purified T4moH from pT4moABEF (1.9 mg·g -1 of cell paste) was typically only 30% of that from pKM11 (6.2 mg·g -1 of cell paste), another pUC18-based vector. More recently, pVP58KABE, a pQE-80 derivative with a Flexi Vector cloning cassette [27,28], has been investigated for the inducible expression of T4moH. This tightly controlled vector gave a yield of purified T4moH greater than 12 mg·g of cell paste [N. Elsen and B. G. Fox, unpublished results].…”

Section: Expression Contextmentioning

confidence: 99%

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Bailey

Elsen

Pierce

et al. 2008

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Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin-and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded ∼1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitiative EPR spectroscopy showed ∼1 mol of the S = ½ signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K M -and apparent k cat -values of 0.15 μM and 3.0 s -1 , respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.