Help on Prokayrotic Protein Expression

Geoffrey Kidd gkidd at aptagen.com
Thu Oct 15 12:55:22 EST 1998


To follow up on Chen Ho An's and Ricardo Silva's comments about codon usage,
here are some references on the subject.  Basically the articles give examples
in which optimizing codons for expression in a particular host species gave
large increases in the amounts of protein expressed.  Codon optimization
requires synthesizing the gene from scratch, which is a service I perform at
Aptagen.  I would be happy to answer any further questions you may have.  Here
are the references:

     1.   Hale RS.  Protein Expr Purif 12:185-8 (1998).  Codon optimization of
the gene encoding a domain from human type 1 neurofibromin protein results in
a threefold improvement in expression level in Escherichia coli.
     2.   Kim CH; Oh Y; Lee TH.  Gene 199:293-301 (1997).  Codon optimization
for high-level expression of human erythropoietin (EPO) in mammalian cells.
     3.   Deng T.  FEBS Lett 409(2):269-72 (1997).  Bacterial expression and
purification of biologically active mouse c-Fos proteins by selective codon
optimization.
     4.   Cormack BP; Bertram G; Egerton M; Gow NA; Falkow S; Brown AJ.
Microbiology 143:303-11 (1997).  Yeast-enhanced green fluorescent protein, a
reporter of gene expression in Candida albicans.
     5.   Prapunwattana P; Sirawaraporn W; Yuthavong Y; Santi DV.  Mol Biochem
Parasitol 83:93-106 (1996).
     6.   Pikaart MJ; Felsenfeld G.  Protein Expr Purif 8:469-75 (1996).
Expression and codon usage optimization of the erythroid-specific
transcription factor cGATA-1 in baculoviral and bacterial systems.
     7.   Yang TT; Cheng L; Kain SR.  Nucleic Acids Res 24:4592-3 (1996).
Optimized codon usage and chromophore mutations provide enhanced sensitivity
with the green fluorescent protein.
     8.   Gouka RJ; Punt PJ; Hessing JG; van den Hondel CA.  Appl Environ
Microbiol 62:1951-7 1996.  Analysis of heterologous protein production in
defined recombinant Aspergillus awamori strains.
     9.   Altmann SW; Timans JC; Rock FL; Bazan JF; Kastelein RA.  Protein
Expr Purif 6:722-6 (1995).  Expression and purification of a synthetic human
obese gene product.
     10.  Kane, J., Current Opinion in Biotechnology 6:494-500 (1995).
Effects of rare codon clusters on gene expression in Escherichia coli.
     11.  Airenne KJ; Sarkkinen P; Punnonen EL; Kulomaa MS.  Gene, 144:75-80
(1994).  Production of recombinant avidin in Escherichia coli.
     12.  Wang, B.Q., L. Lei, and Zachary F. Burton, Protein Expression and
Purification 5:476- 485 (1994).  Importance of codon preference for production
of human RAP74 and reconstitution of the RAP30/74 complex.
     13.  Gerchman SE; Graziano V; Ramakrishnan V.  Protein Expr Purif
5:242-51 (1994).  Expression of chicken linker histones in E. coli: sources of
problems and methods for overcoming some of the difficulties.

AKi Hoji wrote:

> Hi All,
>
> I cloned the cDNA encoding 40kd protein in pET23a, and have tried to
> produce this protein in BL21(DE3) upon IPTG induction, which should
> accumulate in inclusion bodies (for refolding purpose later).  However, I
> have been getting very little expression (compared to my positive control)
> even for 3-4 hrs induction at 0.4mM IPTG.  This protein is  reported to be
> non-toxic to bacteria, and in fact they appear to look fine after
> induction.  I appreciate any suggestions at this point.  Thanks in
> advance.
> --
> Aki Hoji (akhst7+ at pitt.edu)
> Dept. Infectious Diseases & Microbiology
> University of Pittsburgh






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