Question

Basic principles of heterologous cloning and expression

Answer 1

A comprehensive survey of commercially available expression vectors has recently been published [4]. The most commonly used vectors are fusion systems that link additional amino acid sequences (tags) to the protein through a recognition site for a specific protease. Tags may consist of a short peptide sequence or a full protein which can be cleaved from the protein when desired. Presence of tag sequences facilitates solubility, purification, quantification, identification, localization, and assaying of the expressed protein. Frequently used fusion partners include glutathione-S-transferease (GST), his-tag (poly-histidines), maltose binding protein (MBP), thioredoxin (TrxA), FLAG epitope-tag, c-Myc epitope-tag, disulfide isomerase I (DsbA), polyarginine-tag (Arg-tag), calmodulin-binding peptide, cellulose-binding domain, poly-histidine affinity tag (HAT-tag), N-utilizing substance-A (NusA), S-tag, streptavidin-binding peptide (SBP-tag), strep-tag, fluorescent proteins (e.g., green fluorescent protein (GFP)) and ubiquitin [4]. MBP and NusA are specifically used to increase the solubility. MBP is considered to be much more effective for enhancing solubility than GST and thioredoxin [5]. The major disadvantages of fusion protein systems are the requirement of expensive proteases for cleavage from the recombinant protein and the low yield of cleavage reactions [6].

Depending on the host system, vectors for transient or stable expression can be chosen as indicated below.