Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.
DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.
Okamura-Ikeda
K
K
Institute for Enzyme Research, University of Tokushima, Japan.
Fujiwara
K
K
Motokawa
Y
Y
D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019877:Hydroxymethyl and Formyl Transferases; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010449:Peptide Mapping; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification
D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase