A universal expression-purification system based on the coiled-coil interaction of myosin heavy chain.
We have constructed a series of Escherichia coli expression vectors that produce high yields of fusion proteins containing the C-terminal fragment of light meromyosin (LMM) from rabbit fast skeletal muscle. The fusion proteins retain the ability of LMM to form polymers in low salt and to be soluble in high salt. Thus they can be easily purified from bacterial extracts with a high salt-low salt extraction procedure and still retain their biochemical properties. We demonstrate the utility of this system for the heterologous production and simple purification of LMM fusions of p21H-ras, the neurofibromatosis type I protein and the Tat and protease proteins of HIV-1.
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, FRG.
V
V
Wolber
K
K
Maeda
R
R
Schumann
B
B
Brandmeier
L
L
Wiesmüller
A
A
Wittinghofer
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics; D009218:Myosins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D025542:Neurofibromin 1; D015689:Oncogene Protein p21(ras) / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011108:Polymers; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D015696:Gene Products, tat; D015879:Myosin Subfragments; D025542:Neurofibromin 1; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016333:HIV Protease; D009218:Myosins; D015689:Oncogene Protein p21(ras)