Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3). Export

@article{citeulike:2673634, abstract = {Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging.}, address = {Protein Engineering Section, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.}, author = {Kapust, R. B. and Routzahn, K. M. and Waugh, D. S.}, citeulike-article-id = {2673634}, citeulike-linkout-0 = {http://dx.doi.org/10.1006/prep.2001.1545}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/11812224}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=11812224}, doi = {10.1006/prep.2001.1545}, issn = {1046-5928}, journal = {Protein expression and purification}, keywords = {cleavage, cloning, ecoli, expression, protease}, month = {February}, number = {1}, pages = {61--70}, posted-at = {2008-04-15 15:46:12}, priority = {2}, title = {Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3).}, url = {http://dx.doi.org/10.1006/prep.2001.1545}, volume = {24}, year = {2002} }

TY - JOUR ID - citeulike:2673634 L3 - citeulike-article-id:2673634 N2 - Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging. IS - 1 JF - Protein expression and purification SN - 1046-5928 AD - Protein Engineering Section, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA. EP - 70 TI - Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3). VL - 24 SP - 61 KW - cleavage KW - cloning KW - ecoli KW - expression KW - protease AU - Kapust, RB AU - Routzahn, KM AU - Waugh, DS PY - 2002/02// UR - http://dx.doi.org/10.1006/prep.2001.1545 ER -