Boronic acid lectin affinity chromatography (BLAC). 3. Temperature dependence of glycoprotein isolation and enrichment.
In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5-65 degrees C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 degrees C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 degrees C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.
Olajos
Marcell
M
Horváth Laboratory for Bioseparation Sciences, University of Debrecen, 4032 Debrecen, Hungary.
Szekrényes
Akos
A
Hajos
Peter
P
Gjerde
Doug T
DT
Guttman
András
A
D000818:Animals; D001897:Boronic Acids / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006023:Glycoproteins / Q000302:isolation & purification*; D007474:Ion Exchange; D037102:Lectins / Q000737:chemistry; D009113:Muramidase / Q000302:isolation & purification; D009211:Myoglobin / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification; D013696:Temperature; D014361:Trypsin Inhibitors / Q000302:isolation & purification; D014909:Wheat Germ Agglutinins / Q000737:chemistry
D001897:Boronic Acids; D006023:Glycoproteins; D037102:Lectins; D009211:Myoglobin; D014361:Trypsin Inhibitors; D014909:Wheat Germ Agglutinins; D012260:Ribonucleases; C029600:ribonuclease B; D009113:Muramidase