How Much Do You Know About the Prokaryotic Expression System?

E. coli protein expression system is the most commonly used expression system for recombinant proteins. Prokaryotic expression vectors generally consist of the replicon, promoters, selection markers, multiple cloning sites (MCS) and fusion tags / restriction sites.

The replicon is comprised of the origin of replication (ORI) and all of its control elements. The ORI is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive within cells. The best choice of ORI depends on how many plasmid copies you want to maintain, which host or hosts you intend to use, and whether or not you need to consider your plasmid's compatibility with one or more other plasmids. Commonly used pET (Novagen) series vectors mainly carry pMB1 ori, providing a copy number of 15-60; pQE vectors (Qiagen) carrying ColE1 ori provides a copy number of 15-20; pACYC and pBAD series of vectors carry p15A ori, giving a copy number of 10-12, and are commonly used for the co-expression of two recombinant proteins. (Misunderstanding: Does high copy number of the carrier result in high protein expression yield? High copy of the exogenous plasmids will create a greater burden on the normal metabolism of the host, which may slow down cell proliferation, destabilize the plasmid, and eventually compromise the final expression yield of the recombinant protein.) This following table defines common cloning vectors, their copy number, ORI, and incompatibility groups.

Table 1. Common Prokaryotic Expression Vectors and ORIs

Note the A -C compatibility grouping is an arbitrary designation, and plasmids from the same incompatibility group should not be co-transformed.

* Requires pir gene for replication.

**F1 is a phage-derived ORI that allows for the replication and packaging of ssDNA into phage particles. Plasmids with phage-derived ORIs are referred to as phagemid

Promoter

Refers to the DNA sequence that is specific for RNA polymerase’s recognition and binding. Table 2 illustrates a few of the most common prokaryotic expression promoters.

Table 2. Common Prokaryotic Expression Promoters

election Marker

Prokaryotic expression system often uses antibiotics for screening. Commonly used antibiotics are ampicillin, kanamycin, tetracycline, chloramphenicol, etc.

Fusion Tags

Protein fusion tags can be roughly divided into affinity tags and solubility tags. Affinity tags are used primarily for protein purification by affinity chromatography. Solubility tags are often incorporated to promote proper protein folding and enhance solubility. Table 3 and Table 4 list some of the common affinity tags and solubility-promoting tags.

Table 3. Common Affinity Tags

Table 4. Common Solubility Tags

Label Removal

Protein tags are usually removed by enzymatic cleavage after the corresponding affinity purification or dissolution promotion is completed, as the presence of the tag may interfere with the normal function of the target protein. The tag and the recombinant protein are then separated by an affinity column, which simultaneously removes the recombinant endoprotease that often carries an affinity tag as well. N-terminal tags are generally easy to remove while the complete removal of the C-tag is more difficult because most proteases cleave only at the C-terminus of the recognition sequence. Table 5 lists some of the commonly used proteases and their restriction sites.

Table 5. Common Protaminase

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