The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.
The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.
Wingfield
P T
PT
Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892-2775, USA. pelpw@helix.nih.gov
Stahl
S J
SJ
Kaufman
J
J
Zlotnick
A
A
Hyde
C C
CC
Gronenborn
A M
AM
Clore
G M
GM
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding
D015700:HIV Envelope Protein gp41