Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach

Abstract

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of autotrophic organisms. We have constructed a spinach leaf cDNA library in the phage expression vector, lambda gt11, and used a rabbit polyclonal antibody raised against spinach PRK to identify PRK clones. Analyses of the nucleotide sequences of two antibody-positive clones, 1.47 and 1.35 kb in length, showed that they encode a protein which contains the N-terminal amino acid (aa) sequence [Porter et al., Arch. Biochem. Biophys. 245 (1986) 14-23] of mature spinach PRK. The codon for the N-terminal serine of the mature protein occurs 170 bp from the 5' end of the open reading frame (ORF), suggesting that PRK is synthesized with a rather long transit peptide which is removed from the mature enzyme. The ORF, ending with an amber (TAG) codon at position 1054, predicts a mature enzyme of 351 aa with a calculated Mr of 39232.