Structural characterization of recombinant human CD81 produced in Pichia pastoris.
Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom.
Mohammed
M
Jamshad
Sundaresan
S
Rajesh
Zania
Z
Stamataki
Jane A
JA
McKeating
Timothy
T
Dafforn
Michael
M
Overduin
Roslyn M
RM
Bill
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D010843:Pichia / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility / Q000187:drug effects; D012996:Solutions; D060225:Tetraspanin 28; D014461:Ultracentrifugation; D014759:Viral Envelope Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554798:CD81 protein, human; D003902:Detergents; D005960:Glucosides; D011994:Recombinant Proteins; D012996:Solutions; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; C018619:octyl-beta-D-glucoside