Christopher B. Glascock

Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the β- and di-α-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNALeu, similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli.

Transcription of the lac and the hybrid tac promoters is repressed by the lac repressor and induced by the non-metabolizable substrate IPTG. The degree of repression depends upon the ratio of LacI molecules in a cell to the DNA operator sites. In the absence of an inducer, repression of Ptac on a high-copy-number (hcn) plasmid was equivalent in strains containing lacIQ1 on the chromosome, or lacI+ on the plasmid, but not from strains with lacI+ or lacIQ only on the chromosome. Induction of Ptac on hcn plasmids in strains in which expression was controlled by lacIQ1 occurred at very low inducer concentrations (3-10microM IPTG) and reached levels significantly higher than in strains with lacI+ on the plasmid. Greater than 300-fold induction of a beta-LacZ fusion was observed, and >600-fold induction was estimated from recombinant hemoglobin synthesis. Transcription from PlacIQ1 initiated in the same point as PlacI+, but was 170-fold stronger, consistent with the lac repressor levels required to control LacI-regulated genes on hcn plasmids. The DNA sequence upstream of lacI was used to develop a simple PCR test to identify lacIQ1 by a characteristic 15-bp deletion. This deletion created a consensus -35 hexamer, responsible for the increased lacI transcription, and was easily detectable in a variety of strains. Using lacIQ1 hosts eliminates the requirement to maintain lacI on the plasmid to regulate gene expression on hcn expression plasmids.