Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin.
Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of alpha and beta globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.
Gilbert
S C
SC
Delta Biotechnology Ltd, Nottingham, U.K.
van Urk
H
H
Greenfield
A J
AJ
McAvoy
M J
MJ
Denton
K A
KA
Coghlan
D
D
Jones
G D
GD
Mead
D J
DJ
D005822:Genetic Vectors; D006454:Hemoglobins / Q000096:biosynthesis*; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D006454:Hemoglobins; D011994:Recombinant Proteins