There may be three reasons

1. nonspecific binding of other proteins to the amylose matrix (You can find this out by running cell lysage without cloned gene)

2. degradation of the purified protein. This rarely happens. Sometimes, the proteases also contribute for this since you are getting clear bands.

3. truncation. if your lysate is from a mix of different colonies, you may expect this. However, this is also rare. (try different clones)

washing buffer should added extra maltose, if it already contains, increase the amount of maltose or if it is not in buffer add maltose little extra. Avoid external contamination , try to maintain 4 c tem. or again transformed in new bacterial cell with new extracted cloned DNA and confirmed clone.

Hi there

• What did your lysate look like before it was applied to the column
• I agree with the above answers in general terms:
• Try to express your protein from your vector in baterial cells, make a simple lysate and run on an SDS PAGE gel: That way you can asceratin if your protein is being internally processed within your bacerial host: For complex constructs which are alien to the bacteria this is not uncommon
• If you do see this and you are not already attempting to express from a Rec A negative strain like BL 21 De 3 or Rosetta then transform a new host strain with such a genotype
• In addition, vary the expression temp that you induce at and also the Medium you grow your cells in during induction:
• For simple identification of the full length protein heat shock @ 60C and induction for 1 hour in LB agar or an enriched medium like 2 x TY will suffice
• However, for induction of large quantities of protein for a down stream functional assay then a simpler medium plus maltose and prolonged induction at lower temp for prolonged periods e.g. 20C for > 10 hours will often work better in my experience