Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions.
Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.
Teske
Christopher A
CA
Institut für Biotechnologie 2, Forschungszentrum Jülich, 52425 Jülich, Germany.
Simon
Robert
R
Niebisch
Axel
A
Hubbuch
Jürgen
J
D002852:Chromatography, Ion Exchange / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes; D009113:Muramidase