Chromatographic separation of low-molecular-mass recombinant proteins and peptides on Superdex 30 prep grade.
The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30-60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown.
Joyce
J G
JG
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486.
Cook
J C
JC
Przysiecki
C T
CT
Lehman
E D
ED
D000595:Amino Acid Sequence; D060829:Arthropod Proteins; D002845:Chromatography; D002850:Chromatography, Gel; D003911:Dextrans; D006146:Guanidines; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010316:Particle Size; D010455:Peptides / Q000302:isolation & purification*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification
D060829:Arthropod Proteins; D003911:Dextrans; D006146:Guanidines; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide; D012685:Sepharose