A multifunctional vector system for heterologous expression of proteins in Escherichia coli. Expression of native and hexahistidyl fusion proteins, rapid purification of the fusion proteins, and removal of fusion peptide by Kex2 protease.
Vectors have been constructed for the general purpose of expressing foreign proteins in E. coli. These vectors allow the production in high yield of either native proteins or of fusion proteins which contain, at their amino terminus, the peptide Met Gly His6 Ser Gly Leu Phe Lys Arg/, where Leu Phe Lys Arg/ is the recognition site for Kex2 protease which cleaves at the site indicated by /. The His6 sequence is used as a ligand for the one-step affinity purification of the expressed proteins on columns containing Ni or Zn ions chelated to iminodiacetic acid-agarose. After affinity chromatography, the purification peptide is cleaved off with Kex2 protease from Saccharomyces cerevisiae. The vectors also allow site-directed mutagenesis and sequencing of the cloned gene to be expressed without any intermediate subcloning. For practical examples of over-expression, affinity purification, and removal of the purification peptide, we chose a high-molecular-weight protein, phospholipase C gamma 1 (PLC gamma 1, M(r) 148,000) and a low-molecular-weight protein, Hit-1 (M(r) 16,000). Both were obtained pure and in high yield. PLC gamma 1 was fully active; the function of Hit-1 is not known. A set of companion vectors for co-expression of additional proteins has also been developed. These allow expression of proteins which enhance the production or activity of the protein of primary interest and of proteins which exhibit trans-interactions.
Ghosh
S
S
Biochemistry Department, Brandeis University, Waltham, MA 02254, USA.
Lowenstein
J M
JM
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006867:Hydrolases; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010957:Plasmids; D043484:Proprotein Convertases; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004279:DNA, Viral; D007527:Isoenzymes; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C097552:histidine triad protein; D012321:DNA-Directed RNA Polymerases; D006867:Hydrolases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae