Growth kinetics of Escherichia coli and expression of a recombinant protein and its isoforms under heat shock conditions.
Preinduction culture conditions were found to have significant impact on the expression and post-translational modification of a recombinant human protein in Escherichia coli under heat shock conditions (30 to 42 degrees C shift). Higher preinduction growth rates (micrograms) favored better cell viability, greater cell mass yields, and increased cloned gene expression during induction. Formation of recombinant protein isoforms (those containing N epsilon-modified lysine residues) exhibited an increasing trend with increasing micrograms. The different extents of post-translational modifications were suspected to be linked to the different concentrations of certain heat shock protein chaperones resulting from different micrograms. In view of the extensive involvement of E. coli heat shock proteins in cellular activities-including the synthesis, processing, modification, and degradation of proteins-at elevated temperatures, it is believed that micrograms dictated the cellular resources available for synthesizing the heat shock proteins required for cell survival, which in turn determined the ability of the cells to respond to the heat shock. With a higher micrograms both the synthesis of host proteins (as indicated by cell growth and survival) and the cloned gene expression were enhanced. The results demonstrate that there exists an intermediate micrograms for optimum production of the unmodified foreign protein in a heat shock environment. More importantly, they also illustrate the feasibility of improving the recombinant protein homogeneity in fermentation, thereby facilitating downstream processing.
Ryan
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W
Amgen, Inc., Thousand Oaks, California 91320, USA. wen.ryan@amgen.com
Collier
P
P
Loredo
L
L
Pope
J
J
Sachdev
R
R
D019149:Bioreactors; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D015870:Gene Expression; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D007536:Isomerism; D008239:Lysine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature
D011994:Recombinant Proteins; D008239:Lysine