pRK1043

E. coli strain BL21(DE3)-RIL/pRK1043

Description: This derivative of E. coli BL21(DE3) overproduces the catalytic domain of tobaco etch virus (TEV) protease in the form of an affinity sandwich, with E. coli maltose-binding protein fused to its N-terminus and a polyarginine tag fused to its C-terminus. TheTEV protease produced by this strain is the autoinactivation-resistant mutant S219V.

Plasmids: The TEV protease expression vector pRK1043
The tRNA accessory plasmid pRIL (Stratagene)

Replicon: ColE1/pBR322 (pRK1043)
p15A (pRIL)

Antibotic Resistance: Ampicillin (pRK1043)
Chloramphenicol (pRIL)

Promotor: tac (pRK1043)

Inducer: IPTG (1 mM)

Notes: The pRIL plasmid (Stratagene) improves the yield of TEV protease by increasing the supply of argU tRNA in the cell (for AGG and AGA codons). The catalytic activity of the S219V mutant is approximately 2-fold greater than that of the wild-type protease. Shifting the temperature from 37 °C to 30 °C during induction maximizes the yield of soluble MBP-TEV-Arg fusion protein.

Literature Citations: Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000; Kapust, R. B. and Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8: 1668-1674.

Nucleotide Sequence:

Amino Acid Sequence:

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI IFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIK DVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTNSSSNNNNNNNNNNLGIEGRESLFKGPRDYNPISSTICHLTNESDGHTTSLYG IGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPF PQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSP LVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGH KVFMVKPEEPFQPVKEATQLMNRRRRR

Description:	This derivative of E. coli BL21(DE3) overproduces the catalytic domain of tobaco etch virus (TEV) protease in the form of an affinity sandwich, with E. coli maltose-binding protein fused to its N-terminus and a polyarginine tag fused to its C-terminus. TheTEV protease produced by this strain is the autoinactivation-resistant mutant S219V.

Plasmids:	The TEV protease expression vector pRK1043 The tRNA accessory plasmid pRIL (Stratagene)

Replicon:	ColE1/pBR322 (pRK1043) p15A (pRIL)

Antibotic Resistance:	Ampicillin (pRK1043) Chloramphenicol (pRIL)

Promotor:	tac (pRK1043)

Inducer:	IPTG (1 mM)

Notes:	The pRIL plasmid (Stratagene) improves the yield of TEV protease by increasing the supply of argU tRNA in the cell (for AGG and AGA codons). The catalytic activity of the S219V mutant is approximately 2-fold greater than that of the wild-type protease. Shifting the temperature from 37 °C to 30 °C during induction maximizes the yield of soluble MBP-TEV-Arg fusion protein.

Literature Citations:	Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000; Kapust, R. B. and Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8: 1668-1674.

Nucleotide Sequence: