|Unable to clone an insert into the pMAL vector
||Subcloning difficulties or fusion protein expression is toxic to E. coli
||The tac promoter induction ratio of the pMAL plasmid is ~1:30. If the induced level of the fusion is 40% of the total cellular protein, the uninduced level will be over 1%. Try to reduce the amount of protein as this can be toxic, either because of its function (e.g., a protease) or because of its general properties (e.g., very hydrophobic).
|Low yield of pMAL DNA from plasmid preps
||The pMAL plasmid is pBR322-low copy number, but the yield from plasmid preps is often lower than what can be obtained from pBR322
||Modification of the standard alkaline lysis protocol can increase the yield: increasing the volume of the buffers by 1.5-fold doubles the yield of plasmid. For example, for a 500 ml culture, resuspend the cell pellet in 15 ml instead of the standard 10 ml, and increase the denaturing and neutralizing buffer amounts proportionately.
|Following analysis of the fusion protein by Western blot, only a small fraction of the protein is full-length, while most of it migrates close to the MBP6* marker.
||The fusion protein is degraded, leaving a stable MBP-sized breakdown product
||Try using a protease deficient host such as NEB Express, which is Lon- and OmpT-. Additionally, try adding a protease inhibitor cocktail to the lysis buffer.
|Fusion protein is insoluble
||The fusion protein may be misfolded due to rapid protein synthesis at 37°C
||Try expressing at a lower temperature, as low as 15°C. The cells will grow slowly at lower temperatures, so grow the culture at 37°C and shift to the low temperature when adding IPTG. One must also increase the time of induction to compensate for the slower growth–a rule of thumb is 2X for every 7°C.
|When running the uninduced and induced crude extracts on SDS-PAGE side by side, the induced band is not visible.
||Some foreign genes are poorly expressed in E. coli, even when fused to a highly expressed carrier gene.
||Possible explanations are message instability or problems with translation–sometimes it is due to the presence of multiple rare codons in the gene of interest, and in these cases overexpression of the corresponding tRNA can help. Even in cases where a band is not visible, one can get yields up to 5 or 6 mg/liter of culture.
|I’ve cloned my insert, but after SDS-PAGE the only induced band present is the size of MBP6*.
||If the protein of interest is in the wrong translational reading frame, an MBP6-sized band will be produced by translational termination at the first in-frame stop codon.
||The best way to distinguish between these possibilities is to run a Western blot using Anti-MBP Monoclonal Antibody (NEB #E8032). If proteolysis is occurring, at least a small amount of full-length fusion can almost always be detected. DNA sequencing of the fusion junction will confirm a reading frame problem. If the problem is proteolysis, you might want to try NEB Express (NEB #C2523) which is a protease deficient strain lacking the Lon and OmpT proteases.
|If the protein of interest is very unstable, an MBP6-sized breakdown product is usually produced
|The fusion protein flows through the amylose column.
||A factor in the extract may interfere with binding, such as non-ionic detergents and cellular components that are released during alternative methods of lysis.
||Avoid non-ionic detergents, and prolonged treatment with lysozyme or multiple passes through a French press.
|Low intrinsic affinity caused by an interaction between the protein of interest and MBP that either blocks or distorts the maltose-binding site.
||Try shortening or lengthening the polypeptide that is fused to MBP. Alternatively, because the MBP possesses an N-terminal hexahistidine tag, it is possible to purify the MBP-fusion via Immobilized Metal Affinity Chromatography (IMAC).
|Cells grown in LB and similar media have substantial amounts of an amylase that interferes with binding, presumably by either cutting the fusion off the column or by releasing maltose that elutes the fusion from the column.
||By including glucose in the media, expression of this amylase is repressed, and the problem is alleviated.
|The intact fusion protein is detectable by SDS-PAGE, but when I check the crude extract the fusion is degraded.
||For fusions expressed in the cytoplasm, most of the degradation happens during harvest and lysis.
||Harvesting promptly and lysing the cells quickly may help.
|Degradation occurs when the fusion protein is exposed to periplasmic or outer membrane proteases
||Use a host which is deficient in the offending protease(s) and include a protease inhibitor cocktail during lysis
|SDS-PAGE analysis of the purified fusion protein results in multiple bands instead of a single band of the expected MW
||The fusion protein is unstable, which most often leads to degradation in vivo
||One would expect to see bands between the size of MBP (45.5 kDa) and the size expected for the full-length fusion, since fragments smaller than MBP would not bind to the affinity column. An exception would be if the fusion protein breaks down at the junction between MBP and the protein of interest, and the protein of interest oligomerizes. In this situation, the protein of interest may bind to the fusion protein, and therefore a band the size of the protein of interest can appear even if it is smaller than MBP.
|The protein of interest is binding non-specifically to other E. coli proteins, e.g., it has a surface that binds other proteins by electrostatic or hydrophobic interactions.
||Modifications to the column buffer can sometimes be used to help wash the interacting proteins away. Electrostatic interactions can be weakened by including up to 1 M NaCl in the column buffer, and hydrophobic interactions can be weakened by lowering the salt to 25–50 mM NaCl and including 5% ethanol or acetonitrile in the column buffer. Non-ionic detergents (<0.1 %) can also be used to weaken hydrophobic interactions, but they can interfere with the affinity of certain fusion proteins.
|Poor fusion protein cleavage with TEV Protease.
||The fusion protein may be folded in a way that the TEV site is inaccessible
||Anything that perturbs the structure might uncover the site, such as denaturing and refolding or addition of a chaotropic reagent (up to 2 M urea, up to 3 M guanidine HCl, or up to 0.5% SDS). Or try adding amino acid residues to the N-terminus of the protein; either by cloning into one of the downstream sites in the polylinker, or by adding codons to the insert (e.g., adding four alanine codons before the start of the gene). With this strategy, the extra residues remain at the N-terminus after TEV Protease cleavage.