A novel method for quantitative analysis of recombinant secreted proteins using two-dimensional electrophoresis.
We describe a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based approach for detecting and quantifying secreted recombinant proteins in media conditioned by baby hamster kidney (BHK) cells. Seven secreted proteins were analyzed in this system: leptin, thrombopoietin, thrombin, glycoprotein 130, soluble interleukin-2 receptor, and two novel sequences obtained from sequence database searches. BHK cells transfected with plasmids encoding each of these proteins, and cells transfected with empty plasmids (control cells), were metabolically labeled and the resulting conditioned media were analyzed by 2-D PAGE. Gel images derived from cells expressing recombinant proteins were compared with images from control cells in order to identify spots corresponding to the expressed proteins. All seven of the test proteins were successfully detected using this method. The sensitivity of the system was evaluated by diluting samples derived from high-expressing clones with conditioned media from control cells. The sensitivity of detection was protein-dependent, but recombinant proteins expressed at levels as low as 10 ng/mL could be detected. Quantification of recombinant protein levels was achieved by measuring spot intensities using phosphorimager analysis. The intensity of spots corresponding to recombinant proteins were compared with the spot intensity of an endogenous BHK protein which had been calibrated to a known standard. Estimates of the levels of expressed protein determined using this technique correlated with the levels determined using standard affinity assays. We conclude that this system provides a reliable method for quantifying levels of protein expression when specific assays are unavailable.
Nygaard
S C
SC
ZymoGenetics, Inc., Seattle, WA 98102, USA.
Hart
C
C
De Jongh
K S
KS
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D020738:Leptin; D008562:Membrane Glycoproteins; D014176:Protein Biosynthesis; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000096:biosynthesis; D013926:Thrombopoietin / Q000096:biosynthesis
D020738:Leptin; D008562:Membrane Glycoproteins; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D013926:Thrombopoietin; D013917:Thrombin