Expression of a Brassic napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.
Tsang
Edward W T
EW
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9. Ed.Tsang@nrc.ca
Hu
Zhiyuan
Z
Chang
Qing
Q
McGregor
D Ian
DI
Keller
Wilfred A
WA
D000595:Amino Acid Sequence; D001483:Base Sequence; D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D018698:Glutamic Acid / Q000494:pharmacology; D019751:Intramolecular Transferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014018:Tissue Distribution
D011994:Recombinant Proteins; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; C010627:pyridoxamine phosphate