We are trying to express a lectin in E.coli, which is specific to high mannose glycans. Using akta system and size exclusion column (Sephacryl s-200), we isolated protein though at a pretty low purity.

Here is the strange thing, according to previous literature, the lectin is supposed to bind exclusively to high mannose glycans but not monosaccharide mannose, hence should be inhibited by yeast mannan in a hemagglutination inhibition test. What we have found is that adding yeast mannan gives a higher hemagglutination tier... And it takes way much longer for the diluted protein to sink.

Lectin is in 20mM PBS with 0.3M NaCl. Rabbit erythrocyte 2% is used, treated with trypsin and stabilized with 0.2% BSA. Yeast mannan is prepared as a solution in 20mM PBS at concentration of 20mg/ml or 1g/ml (both failed...). Hemagglutination inhibition test was conducted by adding 25ul protein to 25ul yeast mannan solution and finally 25ul of blood was added after 1h incubation at room temperature. 

Can someone help us out please? Thanks!