Modification of the carboxy-terminal amino acid sequence alters the Escherichia coli expression of a gene encoding multiple repeats of a bovine growth hormone releasing factor analog

Since investigations into the determinants of intracellular protein degrada tion have shown that the carboxy terminal sequence can be a critical factor for protein expression in Escherichia call, we attempted to increase the e xpression of a protein containing multiple repeats of a bovine growth hormo ne releasing factor analog (bGRF30) by modifying the carboxy terminus with the addition of short amino acid extensions derived from stable E. coli pro teins. Extensions capable of increasing bGRF30 per liter titers up to four- fold, as well as extensions that completely abolished bGRF30 expression wer e identified. Select C-terminal extensions were investigated further to det ermine the mechanism by which they affected bGRF30 expression. Analysis of mRNA levels and protein production titers suggests that extensions which in crease bGRF30 titers primarily affect protein stability and ribosomal relea se. Negative extensions exert their influence through a more complex mechan ism, appearing to interfere with the ability of ribosomes to be efficiently released from their cognate mRNA. (C) 2000 Elsevier Science B.V. All right s reserved.