We report here the development of a thermal responsive affinity ligand specific to sialic
acid, sialic acid containing oligosaccharides, glycoproteins, and other sialylated glycoconjugates.
The ligand is a fusion protein of 40 repeats of pentapeptide of an elastin like polymer (ELP) and the
21 kD sialic acid binding domain of a Vibrio cholera neuraminidase (VCNA). For cost-effective
synthesis, the fusion protein was targeted to the periplasmic space of an E. coli lpp deletion mutant,
resulting in its secretion to the growth medium. A pre-induction heat-shock step at 42 ˚C for 20
minutes was necessary to achieve high level expression of the ligand. Under optimized induction
condition (18 ˚C, 0.1 mM IPTG and 48... More
We report here the development of a thermal responsive affinity ligand specific to sialic
acid, sialic acid containing oligosaccharides, glycoproteins, and other sialylated glycoconjugates.
The ligand is a fusion protein of 40 repeats of pentapeptide of an elastin like polymer (ELP) and the
21 kD sialic acid binding domain of a Vibrio cholera neuraminidase (VCNA). For cost-effective
synthesis, the fusion protein was targeted to the periplasmic space of an E. coli lpp deletion mutant,
resulting in its secretion to the growth medium. A pre-induction heat-shock step at 42 ˚C for 20
minutes was necessary to achieve high level expression of the ligand. Under optimized induction
condition (18 ˚C, 0.1 mM IPTG and 48 hours of post-induction cultivation), the ligand was produced
to about 100 mg/L. The ligand exhibited a transition temperature of 52 ˚C, which could be depressed
to 37 ˚C with the addition of 0.5 M NaCl. Using fetuin as a model sialylated protein, the ligand was
applied in an affinity precipitation process to illustrate its potential application in glycoprotein
isolation. The ligand captured 100% fetuin from an aqueous solution when the molar ratio of ligand
to fetuin was 10 to 1, which was lower than the expected for full titration of sialic acid on the
glycoprotein by the lectin. Elution of fetuin from ligand was achieved with PBS buffer containing 2
mM sialic acid. To evaluate how protein and other contaminants influence the recovery of sialylated
proteins, CHO medium was spiked into the fetuin solution. The predominant protein species in CHO
medium was found to be albumin. Although its removal of over 94% was evident, purified fetuin
contained some albumin due to its over-abundance. Additional experiments with albumin
contaminant of varying concentrations showed that below 1 mg/L, albumin had no impact on the
affinity precipitation, whereas above 10 mg/L, some albumin was co-purified with fetuin. However,
even at 50 mg/ml, fetuin recovery yield remained high (about 90%) and about 95% of albumin was
depleted with only one cycle of precipitation and elution. These results suggest that the ligand could be used in initial capture of sialylated proteins and to remove over-abundant albumin contaminant.
To the best of our knowledge, this is the first thermal responsive sialic acid binding protein
developed with gene fusion technology.