Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli.
Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.
Köpke
A K
AK
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Germany.
Hannemann
F
F
Boeckh
T
T
D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000235:genetics*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000138:chemical synthesis; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D000596:Amino Acids; D009841:Oligonucleotides; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12