Expression and purification of recombinant human zona pellucida proteins.
Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described.
Harris
J D
JD
Zonagen Inc., 2408 Timberloch Place, Suite B-4, The Woodlands, Texas 77380, USA. jdh@zonagen.com
Seid
C A
CA
Fontenot
G K
GK
Liu
H F
HF
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002853:Chromatography, Liquid; D006224:Cricetinae; D004527:Egg Proteins / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072216:Zona Pellucida Glycoproteins