Team:Calgary/Xpress
Overview
Xpress is an easy-to-use expression vector for E. coli BL21 system designed by the Schryvers’s lab. With a T7 promoter, this vector is easily inducible with IPTG or lactose. Two unique features set this expression vector apart from the other E. colivectors: a quick screening system allows for fast cloning of genetic constructs into E. coli and a Glutathione S-transferase tag (GST) for easy expression of difficult proteins.
Quick Screening
Screening for positive colonies following cloning is often a long and tedious task that often requires screening of many colonies. With the help of a unique feature on the Xpress vector, screening for a positive colony could be done in one single step. Xpress contains two selection markers: a kanamycin resistance gene and an ampicillin resistance gene. However, upon cloning of the insert into the appropriate spot in the multiple cloning sites, the kanamycin resistance gene will be knocked out. As such, successfully cloned colonies will only be resistant to ampicillin whereas colonies that have not been engineered properly will retain their kanamycin resistance. By streaking colonies on ampicillin and kanamycin plates, successful and unsuccessful colonies could be easily differentiated from one another as the successful colonies are expected to only grow on the ampicillin plates while all other negative colonies will show growth on both kanamycin and ampicillin plate. Thus, in this one simple step Xpress allows for fast screening of positive colonies.
Figure 1. Summary of Xpress.
Ease of Expression
E. coli has been an extremely powerful factory for producing proteins. Heterologous protein expression, however, could sometimes prove challenging. In particular, for proteins that are not suited to the natural expression conditions found in E. colior for insoluble proteins that are difficult to produce. Xpress contains a GST tag which is a protein that is highly soluble and easily produced by E. coli fusion of GST to heterologous proteins has proven to help with their expression as GST can act as a chaperone that helps with the proper folding of the protein [1]. By using the BamHI and HindIII restriction cloning, the insert of interest could be fused to the GST protein, allowing them to be more easily expressed in E. coliThus, with the help of a GST protein, Xpress is the optimal vector for the production of difficult proteins.
Figure 2. Glutathione S-transferase (GST) Protein
References
Harper S, Speicher DW. Purification of proteins fused to glutathione S-tranferase. Methods in molecular biology (Clifton, N.J.). 2011 [accessed 2021 Oct 20];681:259. /pmc/articles/PMC3584333/. doi:10.1007/978-1-60761-913-0_14
