pHKP1212

E. coli strain BL21(DE3)/pHKP1212

Description: This derivative of E. coli BL21(DE3) overproduces the catalytic domain of tobbaco vein mottling virus (TVMV) protease fused to the C-terminus of E. coli maltose-binding protein.

Plasmid: The TVMV protease expression vector pHPK1212, a derivative of pMal-C2 (New England Biolabs)

Replicon: ColE1 (pBR322)

Antibotic Resistance: Ampicillin

Promotor: tac

Inducer: IPTG (1 mM)

Notes: The specificity of TVMV protease is distinct from that of TEV protease. The canonical recognition site for TVMV protease is ETVRFQS, with cleavage occurring between Q and S. Shifting the temperature from 37 °C to 30 °C during induction maximizes the yield of soluble MBP-TVMV protease fusion protein. The TVMV protease open reading frame on pHPK1212 was constructed from synthetic oligonucleotides and has been codon-optimized for expression in E. coli.

Literature Citations: Yoon, H. Y., Hwang, D. C., Choi, K. Y., and Song, B. D. (2000). Proteolytic processing of oligopeptides containing the target sequences by the recombinant tobacco vein mottling virus NIa protease. Mol. Cells 10: 213-219.

Nucleotide Sequence:

Amino Acid Sequence:

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI IFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIK DVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTNSITSLYKKAGSKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYII ANQHLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQ PTIKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGN ILGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWFNADKISWGSFTLVEDAPEDD FMAKKTVAAIMD

Description:	*This derivative of E. coli* BL21(DE3) overproduces the catalytic domain of tobbaco vein mottling virus (TVMV) protease fused to the C-terminus of E. coli maltose-binding protein.**

Plasmid:	The TVMV protease expression vector pHPK1212, a derivative of pMal-C2 (New England Biolabs)

Replicon:	ColE1 (pBR322)

Antibotic Resistance:	Ampicillin

Promotor:	*tac*

Inducer:	IPTG (1 mM)

Notes:	The specificity of TVMV protease is distinct from that of TEV protease. The canonical recognition site for TVMV protease is ETVRFQS, with cleavage occurring between Q and S. Shifting the temperature from 37 °C to 30 °C during induction maximizes the yield of soluble MBP-TVMV protease fusion protein. The TVMV protease open reading frame on pHPK1212 was constructed from synthetic oligonucleotides and has been codon-optimized for expression in E. coli.

Literature Citations:	Yoon, H. Y., Hwang, D. C., Choi, K. Y., and Song, B. D. (2000). Proteolytic processing of oligopeptides containing the target sequences by the recombinant tobacco vein mottling virus NIa protease. Mol. Cells 10: 213-219.

Nucleotide Sequence: