Radical Changes in the Engineering of Synthetic Genes for Protein Expression - - BioPharm International


Radical Changes in the Engineering of Synthetic Genes for Protein Expression

BioPharm International

Extensive alteration of codon usage alone, however, can introduce deleterious pauses into an open reading frame. In addition, changing the host organism effectively randomizes the pauses, making expression unpredictable. Managing codon pair interactions and simultaneously optimizing the entire set of parameters requires advanced computationally intensive design tools. Successful applications to explore and exploit the incredible diversity of potential gene sequences have been developed using a branch–bound algorithm.15 The design process called "translation engineering," for example, simultaneously satisfies the five parameters listed above.12

Continuing use of these techniques for ever-increasing numbers of genes in diverse organisms will further test their utility. To date, genes have been tailored for expression in E. coli, gram positive bacteria, yeasts, human cells, and plants, with frequently dramatic increases in protein yield.

The Future of Translation Engineering and the Role of Pausing in Complex Proteins

A rich series of biological events takes place during the translation of a protein. Deconvoluting the various aspects of co-translational modification critically depends on modulating the speed of the ribosome. One example is the independent domain folding of multi-domain proteins. Experimental evidence shows that proper folding of a yeast TY3 GAG gene (which has two independent domains) expressed in E. coli can be altered by the placement of pause sites between the two domains.16 By modifying the translation kinetics of complex multi-domain proteins, it may be possible to alter the time each domain needs to organize. Although refolding studies indicate that the time required for a protein to settle into its final configuration may take as long or longer that the translation of the protein,17 pausing may allow each domain to partially organize, committing to a particular, independent fold. Other co-translational events, such as association with membranes, secretion, proteolysis, or association with other proteins, may all depend on the kinetics of the emerging nascent peptide.

Evolution has favored the over-representation of codon pairs that encode pause sites. This overabundance presumably is renewed as the set of pause sites change over evolutionary time. It appears that tremendous genome-wide selection pressure exists for cells to avoid creating misfolded proteins.18 Perhaps these codon pair-encoded pauses are selected as a consequence of this pressure. Would-be designers of genes should consider taking advantage of this rich and biologically-used method for controlling the translation of genes and the activity of their encoded proteins.


The authors wish to thank GW Hatfield, RH Lathrop, S-P Hung, and A Fraudorf.

Joseph D. Kittle, Jr, Ph.D., Senior Vice President, Market Development, CODA Genomics, Inc., 4521 Campus Drive, Irvine, CA 92612, Office 949 348 1188 ext 103


1. Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci U S A. 1979; 76:106-10.

2. Swartz JR. Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol. 2001;12:195-201

3. Greene JJ. Host cell compatibility in protein expression, Methods Mol Biol. 2004;267:3-14.

4. Gerngross TU. Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol. 2004;22:1409-14

5. Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant protein expression in Pichia pastoris. Mol Biotechnol. 2000;16:23-52.

6. Andersen DC, Krummen L. Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol. 2002;13:117-23

7. Gutman GA, Hatfield GW Nonrandom utilization of codon pairs in Escherichia coli. Proc. Natl. Acad. Sci. USA 1989; 86: 3699-3703

blog comments powered by Disqus



First Biosimilar Application Kicks Off Legal Battle
October 31, 2014
FDA Approves Pfizer's Trumenba for the Prevention of Meningitis B
October 30, 2014
EMA: Extrapolation Across Indications for Biosimilars a Possibility
October 30, 2014
Bristol-Myers Squibb Announces Agreement to Acquire HER2-Targeted Cancer Treatment
October 29, 2014
Amgen, Sanofi, and Ono Pharmaceuticals Partner with Universities on Transmembrane Protein Research
October 28, 2014
Author Guidelines
Source: BioPharm International,
Click here