Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme.
A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.
Dahl
S W
SW
Unizyme Laboratories A/S, Dr. Neergaards Vej 17, Horsholm, Denmark, swd@unizyme.dk
Slaughter
C
C
Lauritzen
C
C
Bateman
R C
RC
Connerton
I
I
Pedersen
J
J
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase