Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli

• Published in: Biochimie Vol. 73; no. 6; pp. 647 - 655
• Main Authors: Köpke, A.K.E., Hannemann, F., Boeckh, T.
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01.06.1991; Elsevier
• Subjects: Amino Acids - analysis; Archaea - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Chromatography, Ion Exchange; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; gene modifications; Genetic Engineering; Genetic technics; Genetic Vectors; Methods. Procedures. Technologies; Miscellaneous; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotides - chemical synthesis; overexpression; protein purification; ribosomal proteins; Ribosomal Proteins - biosynthesis; Ribosomal Proteins - genetics; Ribosomal Proteins - isolation & purification; Synthetic digonucleotides and genes. Sequencing; ribosomal proteins; protein purification; gene modifications; overexpression; DTE; DMF; Purification; Archaeobacteria; Escherichia coli; HPLC chromatography; Gene expression; Halobacterium halobium; Ribosomal protein; Biological activity; Ion exchange chromatography; Gene; Bacteria; Two dimensional electrophoresis; Mutation; Halobacteriaceae; Enterobacteriaceae
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/0300-9084(91)90044-2

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 eaturing a tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed 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.