Coupled Protein Expression & Purification

It is often desirable to design a method of purification directly into a recombinant protein expression strategy. This is most often accomplished by expressing a target protein with an appended “tag” that permits its purification by affinity chromatography. Tags can be a short peptide that specifically interacts with an immobilized antibody or metal ion, or larger binding domains that interact with specific immobilized ligands.

An important consideration in using a protein-tagging strategy is whether or not the presence of a tag will affect the protein’s downstream applications. For example, the presence of a larger binding domain may permit more simple purification, but it might also adversely affect the biochemical function of the target protein it is fused to. Therefore, it is often desirable to include a method for tag removal following purification of the fusion protein. Common methods include the use of site-specific proteases to cleave at an engineered site between the tag and the target protein, or the use of an intein to auto-catalyze tag removal.

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Coupled Protein Expression & Purification includes these areas of focus:
IMPACT™ Protein Expression
Maltose Binding Protein Expression
His-tagged Protein Expression
Yeast Chitin Binding Domain Tag
FAQs for Coupled Protein Expression & Purification
Protocols for Coupled Protein Expression & Purification
Application Notes Coupled Protein Expression & Purification
Overview of Secretion in K. lactis 
In the nucleus, an integrated expression vector encoding a fusion between the α-MF domain (blue) and a desired protein (black) is expressed. A signal peptide in the α-MF domain directs entry of the fusion protein into the endoplasmic reticulum (ER) and is removed by signal peptidase (SP). The fusion protein is transported to the Golgi where the Kex protease removes the α-MF domain. The protein of interest is then secreted from the cell.
The pKLAC2 Expression Vector
pKLAC2 (9107 bp) contains the 5´ and 3´ ends of the LAC4 promoter (PLAC4-PBI) separated by DNA encoding β-lactamase (ApR) and the pMB1 origin (ori) to allow for its propagation in E. coli. The K. lactis α-mating factor secretion leader sequence (α-MF), multiple cloning site (MCS), and the LAC4 transcription terminator (TT) lie immediately downstream of 3´ PLAC4-PBI. The yeast ADH1 promoter (PADH1) drives expression of an acetamidase selectable marker gene (amdS). The vector can be linearized by digestion with SacII or BstXI to create a linear DNA fragment capable of inserting into the native LAC4 promoter region of the K. lactis genome.
Expression of MBP with K. lactis
Lane 1: Protein Marker, Broad Range (NEB #P7702)
Lane 2: spent culture medium (15 µl) from wild-type K. lactis cells.
Lane 3: spent culture medium (15 µl) from K. lactis cells harboring an integrated expression cassette containing the E. coli malE gene.
pKLAC2 Multiple Cloning Site
pKLAC2 (9107 bp) contains the K. lactis α-mating factor secretion leader sequence (blue background) and a polylinker immediately downstream of the PLAC4-PBI promoter. Unique polylinker restriction sites are indicated. Half arrows show the positions of pKLAC-series vector-specific sequencing primers available from New England Biolabs.
Schematic Illustration of Purification Using pMAL™ System
Protein Expression Using pMAL™
SDS-polyacrylamide gel electrophoresis of fractions from the purification of MBP-paramyosin-ΔSal.
Lane 1: Protein Ladder (NEB #P7703).
Lane 2: uninduced cells.
Lane 3: induced cells.
Lane 4: purified protein eluted from amylose column with maltose.
Lane 5: purified protein after Factor Xa cleavage.
Lane 6: paramyosin fragment in flow-through from second amylose column.
Schematic Illustration of the IMPACT® System
Expression and Purification of E. coli Maltose-Binding Protein (MBP) Using the IMPACT System 
Lane 1: uninduced cell extract.
Lane 2: induced cell extract showing expressed fusion protein.
Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C.
Lane 4: MBP ligated to a peptide containing an N-terminal cysteine.
Marker M is the Protein Ladder (NEB #P7703)
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