Promoters

ATUM uses several different inducible promoter systems. The rhamnose-inducible promoter combines high expression levels with precision of control. The T7 system requires special host cells containing a chromosomal copy of the T7 polymerase under control of the lac promoter. IPTG induction expresses T7 polymerase, which then transcribes any gene downstream of a T7 promoter. The IPTG-inducible T5 promoter consists of a strong constitutive promoter flanked by lac operator sequences and works in any strain of E. coli. The PhoA promoter does not require expensive or metabolizable inducers, but auto-induces once the cells have depleted the phosphate from the media.

Promoter Properties

Different promoters work better for different proteins

Expression using a specific promoter can be further controlled by the copy level of the origin of replication. In particular, uninduced expression levels of toxic proteins can be further reduced by using low copy versions of the vectors.

Signal Sequences

In Gram negative bacteria, proteins tend to be poorly secreted and are often directed to the periplasm. The periplasm provides a different environment to the cytoplasm, most significantly the periplasm is oxidizing and allows disulfide bond formation. During bacterial protein expression, the effectiveness of a signal sequence in directing the protein to the periplasm varies depending upon the protein being expressed.

Effect of different signal sequences on yield and solubility of different proteins directed to the periplasm in E. coli.

Open Reading Frame

Our earliest work in developing our GeneGPS® design platform was performed in E. coli. From E. coli codon optimization, we have subsequently applied GeneGPS® to optimize genes for expression in a range of bacterial hosts including Bacillus and Clostridium. By designing and testing hundreds of genes and modelling the data, we are able to identify the critical sequence parameters that most strongly influence expression. The graph below shows a strong correlation between our gene optimization model predictions and measured gene expression levels: this is the same model that our backtranslation algorithms use to design genes for bacterial protein expression.

GeneGPS algorithms to design genes for high expression in E. coli

Genes encoding fluorescent protein, scFv antibody fragments and Phi29 polymerase optimized for expression in E. coli are shown as filled circles. Open circles show non-optimized gene constructs.

Solubility-Enhancing Fusions

Proteins expressed in bacteria do not always fold well and can aggregate in insoluble inclusion bodies. ATUM uses several different fusion tags during bacterial protein expression to improve the solubility of your proteins. Protease cleavage sites between the tag and your protein allow the removal of the tag from the desired protein product.

Effect of different tags on the yield and solubility of three different proteins produced in E. coli. The His tag is an affinity tag but does not generally affect solubility. It is included here to show the solubility of the proteins in the absence of solubility-enhancing fusions.