looking for argU expression vector.

nigel at nature.berkeley.edu nigel at nature.berkeley.edu
Tue Jul 8 20:35:51 EST 1997


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

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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.



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