Enhancement of recombinant protein synthesis and stability via coordinated amino acid addition.
In this work, effective feeding schemes that would minimize stress responses to cloned-protein overexpression are investigated. The cloned-protein (chloramphenicolacetyl-transferase, CAT) contains a high aromatic amino acid content, most notably a high phenylalanine content. Experiments performed on Escherichia coli RR1 [pBR329] (constitutive promoter) and E. coli JM105 [pSH101] (inducible promoter) demonstrated that phenylalanine addition increases the rate of synthesis and yield of CAT. A previous study correlating inducer strength with CAT expression in E. coli JM105 [pSH101] indicated that the highest expression rate was accompanied by the highest apparent rate of protein degradation. In this work, the combined addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) and phenylalanine at intermediate levels resulted in substantial increase of CAT synthesis and partial reduction of protein degradation. Furthermore, transmission electron micrographs verified the absence of inclusion bodies, which, along with proteases, were suspected to reduce protein activity. The research demonstrates that significant enhancement in production and stability of heterologous proteins is possible by designing feeding strategies that incorporate knowledge of the interaction between primary cellular metabolism and foreign protein expression.
Ramérez
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DM
Center for Agricultural Biotechnology and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
Bentley
W E
WE