Chapter 12 - Expression of recombinant proteins in prokaryotic host

Abstract

Escherichia coli due to its fast growth and ease of genetic manipulation is one of the preferred hosts for the expression of recombinant proteins. The selection of suitable expression system and protocols are prerequisite for the expression of recombinant proteins. For overexpression, the host cells must be transformed with a construct containing the gene of interest followed by the selection and cultivation of the correct construct. Most of the E. coli expression vectors use lac operon. The presence of lac operator (lacO) downstream to the promoter of the target gene inhibits the transcription initiation due to the binding of the lac repressor which is removed after adding lactose or the isopropyl-β-d-thiogalactopyranoside (IPTG). Here our focus is limited to IPTG-inducible vector in E. coli host. The pET vector system is one of the widely used systems for expressing recombinant proteins in E. coli. For the activation of recombinant protein expression, the sufficient amount of inducer i.e., 0.5–1.0mM of IPTG is added to stop the binding of repressor LacI in presence of T7 RNA polymerase within the host cell. The T7 RNA polymerase is capable of transcribing almost any DNA fragment that is under the control of T7 promoter and is about five times faster compared to E. coli RNA polymerase. Once, the system is induced, cell's resources are completely used to express the gene of interest and within hours of induction nearly half of the cell's total protein is contributed by the recombinant protein. The expression of recombinant proteins in the pET expression system can be regulated using concentration of IPTG supplied to host cells. This approach is particularly suitable during the expression of proteins with limited solubility. Moreover, using different types of host for initial cloning and overexpression, the gene of interest can be kept in a transcriptionally silent state when T7 RNA polymerase is not present.