Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system

2012 ◽  
Vol 109 (8) ◽  
pp. 1987-1995 ◽  
Author(s):  
Robert P. Harris ◽  
Jo Mattocks ◽  
Philip S. Green ◽  
Frank Moffatt ◽  
Peter M. Kilby
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Production System ◽  
Recombinant Escherichia Coli ◽  
Low Level ◽  
And Control

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals Cloning of a Novel Gene for Quinolone Resistance from a Transferable Plasmid in Shigella flexneri 2b

2005 ◽  
Vol 49 (2) ◽  
pp. 801-803 ◽  
Author(s):  
Mami Hata ◽  
Masahiro Suzuki ◽  
Masakado Matsumoto ◽  
Masao Takahashi ◽  
Katsuhiko Sato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Shigella Flexneri ◽  
Amino Acid Identity ◽  
Quinolone Resistance ◽  
Acid Identity ◽  
Low Level ◽  
Novel Gene ◽  
Level Resistance

ABSTRACT A novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coli HB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.

scholarly journals A complex mutant of TEM-1 beta-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate.

1997 ◽  
Vol 41 (6) ◽  
pp. 1322-1325 ◽  
Author(s):  
D Sirot ◽  
C Recule ◽  
E B Chaibi ◽  
L Bret ◽  
J Croize ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Direct Sequencing ◽  
Hydrolytic Activity ◽  
Beta Lactamase ◽  
E Coli ◽  
Low Level ◽  
Extended Spectrum ◽  
Extended Spectrum Beta Lactamases ◽  
Synergy Test

Escherichia coli GR102 was isolated from feces of a leukemic patient. It expressed different levels of resistance to amoxicillin or ticarcillin plus clavulanate and to the various cephalosporins tested. The double-disk synergy test was weakly positive. Production of a beta-lactamase with a pI of 5.6 was transferred to E. coli HB101 by conjugation. The nucleotide sequence was determined by direct sequencing of the amplification products obtained by PCR performed with TEM gene primers. This enzyme differed from TEM-1 (blaT-1B gene) by four amino acid substitutions: Met-->Leu-69, Glu-->Lys-104, Gly-->Ser-238 and Asn-->Asp-276. The amino acid susbstitutions Leu-69 and Asp-276 are known to be responsible for inhibitor resistance of the IRT-4 mutant, as are Lys-104 and Ser-238 substitutions for hydrolytic activity of the extended-spectrum beta-lactamases TEM-15, TEM-4, and TEM-3. These combined mutations led to a mutant enzyme which conferred a level of resistance to coamoxiclav (MIC, 64 microg/ml) much lower than that conferred by IRT-4 (MIC, 2,048 microg/ml) but higher than that conferred by TEM-15 or TEM-1 (MIC, 16 microg/ml). In addition, the MIC of ceftazidime for E. coli transconjugant GR202 (1 microg/ml) was lower than that for E. coli TEM-15 (16 microg/ml) and higher than that for E. coli IRT-4 or TEM-1 (0.06 microg/ml). The MICs observed for this TEM-type enzyme were related to the kinetic constants Km and k(cat) and the 50% inhibitory concentration, which were intermediate between those observed for IRT-4 and TEM-15. In conclusion, this new type of complex mutant derived from TEM-1 (CMT-1) is able to confer resistance at a very low level to inhibitors and at a low level to extended-spectrum cephalosporins. CMT-1 received the designation TEM-50.

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