EFFECTS OF CODON USAGE AND VECTOR-HOST COMBINATIONS ON THE EXPRESSIONOF SPINACH PLASTOCYANIN IN ESCHERICHIA-COLI

Spinach plastocyanin has been expressed in Escherichia coli and export ed to the periplasmic space. The effects of codon usage, expression sy stem, growth length, and temperature on expression levels in LB medium were investigated. A stretch of codons, rare in E. coli, was identifi ed and replaced with highly expressed codons, increasing the yield by at least 20%. Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter. Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction. The opt imized expression system produced 38 mg holoprotein per liter culture. In this system it was also possible to express plastocyanin in minima l medium, at a yield of 10 mg per liter. N-terminal sequencing and mas s spectrometry showed that plastocyanin was correctly processed. The e xpressed plastocyanin was purified to homogeneity, as shown by an A(27 8)/A(597) ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorpti on coefficients for epsilon(278) and for epsilon(597) were determined to be 4700 M-1 cm(-1). (C) 1997 Academic Press.