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 beta- and di-alpha-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.
Apostol
I
I
Somatogen, Inc., Boulder, Colorado 80301, USA.
Levine
J
J
Lippincott
J
J
Leach
J
J
Hess
E
E
Glascock
C B
CB
Weickert
M J
MJ
Blackmore
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D006454:Hemoglobins; D011994:Recombinant Proteins; C005313:norvaline; D007930:Leucine; D014633:Valine