Hi,
I've got a protein that's been making my life a nightmare...and I think
I've finally hit on the reason. Apparently I need to give e.coli a little
more argU tRNA so that it can translate it. (I've got three AGG's in a
row, as well as four pairs of AGG AGA/AGG).
Is there a commercial source for an argU overexpression vector/host? Does
anyone out there have a plasmid they'ed care to share?
thanks in advance
-nigel
--
Nigel Walker [>======<] nigel at mendel.berkeley.edu
Dept. of Plant Biology \ Mu / (510) 642-6813 Lab
351 Koshland hall \ / (510) 642-4995 Fax
U.C. Berkeley \ / (510) 763 7862
Berkeley, CA 94720 \/ http://plantbio.berkeley.edu/~nigel
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PS: here's a couple of ref's in case this interests you.
3. (BIOSIS result)
Zahn, K.
Overexpression of an mRNA dependent on rare codons inhibits protein
synthesis and cell growth.
Journal of Bacteriology, v.178, n.10, (1996): 2926-2933.
Abstract:
lambda's int gene contains an unusually high frequency of the rare
arginine codons AGA and AGG, as well as dual rare Arg codons at three
positions. Related work has demonstrated that Int protein expression
depends on the rare AGA tRNA. Strong transcription of the int mRNA with a
highly efficient ribosome-binding site leads to inhibition of Int protein
synthesis, alteration (if the overall pattern of cellular protein
synthesis, and cell death. Synthesis or stability of int and ampicillin
resistance mRNAs is not affected, although a portion of the untranslated
int mRNA appears to be modified in a site-specific fashion. These
phenotypes are not due to a toxic effect of the int gene product and can
be largely reversed by supplementation of the AGA tRNA in cells which
bear plasmids expressing the T4 AGA tRNA gene. This indicates that
depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA
And AGG codons on the overexpressed int mRNA underlies all of these
phenomena. It is hypothesized that int mRNA's effects on protein
synthesis and cell viability relate to phenomena involved in lambda phage
induction and excision.
4. (BIOSIS result)
Hu, X; Shi, Q; Yang, T; Jackowski, G.
Specific replacement of consecutive AGG codons results in high-level
expression of human cardiac troponin T in Escherichia coli.
Protein Expression and Purification, v.7, n.3, (1996): 289-293.
Abstract:
The adult isoform of human cardiac troponin T (TnT) contains 288 amino
acids, 14 of which (4.9%) are encoded by the rarely used arginine codons
(12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient
quantity of TnT protein for antibody production, we cloned the
corresponding cDNA and expressed it in E. coli. A low-level expression of
TnT that comprised only about 1% of total cell protein was initially
observed with the use of the native cDNA. The existence of two pairs of
consecutive AGG codons (AGG-165AGG-166 and AGG-215AGG-216) in the cDNA
was suspected to be the main cause for this low-level expression. These
two pairs of consecutive AGG codons were successively replaced with the
major synonymous codon CGT by site-directed mutagenesis. As suspected, a
10-fold increase in TnT expression was obtained when one pair of the rare
arginine codons was replaced and a 40-fold increase was achieved when
both pairs of the rare codons were replaced. Our finding demonstrates the
importance of consecutive rare codons in the suppression of high-level
expression of heterologous proteins in E. coli and suggests that in order
to maximize protein expression, a similar approach may be taken with
other genes which contain consecutive rare codons.
