Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A.
A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.
Rondahl
H
H
Department of Microbiology, Kabi Pharmacia, KabiGen, Stockholm, Sweden.
Nilsson
B
B
Holmgren
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A