THE CLONING AND EXPRESSION OF TCL

The Cloning and Expression of TCL via Baculovirus in Insect Cells

ABSTRACT

TCL is a member of the Rho-family GTPases, which are responsible for many cellular processes such as cell morphology, motility, and apoptosis. The function of TCL as a GTPase is not well understood. In order to clarify its function, TCL will be expressed and purified in insect cells. TCL was initially cloned into a plasmid using specialized Escheria coli cells, which were subsequently used to transform bacteria containing special bacmid DNA. The bacteria were screened to identify only those that incorporated TCL into the bacmid. The bacteria were lysed and the bacmid was used to transfect insect cells to produced baculovirus, an insect specific virus. The baculovirus with the TCL was used to infect insect cells, which were incubated to produce the TCL protein.

The Cloning and Expression of TCL via Baculovirus in Insect Cells

Introduction

The use of baculovirus remotes to the early 1950s when they were used as natural control agents of insect pest populations. At present, genetically engineered recombinant baculovirus (rBAC) are viewed as powerful vectors for expressing heterologous proteins in insect cells, both in vivo and in vitro. In heterologous protein production, one of the most used processes consists on the infection of Sf-9 cells with AcMNP. This system offers some advantages over prokaryotic and other eukaryotic systems, namely: (a) the very strong polyhedrin promoter with protein expression levels up to 500 mg l-1; (b) ability to express large proteins (>50 kDa); (c) correct post-translation modifications and folding; (d) low cost; (e) the ability to express large cDNA inserts as well as multiple genes.

The Baculovirus Expression Vector System (BEVS) has been widely used in research and scientific industrial communities for the production of high levels (up to 1000mg/mL) of properly post-translationally modified (folding, disulfide bond formation, oligomerization, glycosylation, acylation, proteolytic cleavage), biologically active and functional recombinant proteins. The Baculovirus Expression Vector System is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene (Jensen 2008)

Method

Ligation process

It is the process of annealing or joining, such as the DNA ligase-catalyzed joining of two DNA fragments to form a recombinant molecule. Prior to ligation, compatible insert and vector DNA was prepared. Both need compatible termini and must be free of contaminating chemicals such as phenol, ethylenediaminetetraacetic acid (EDTA) or ethanol that may inhibit ligation. Vector DNA (pFBac2) was prepared by digestion with the relevant restriction enzyme (T4 DNA ligase), then dephosphorylated by incubation with alkaline phosphatase to remove the terminal 5' phosphate groups and so prevent vector self-ligation. (“T4 DNA ligase definition - ExpertGlossary,” n.d.)

Bacterial transformation

E.coli was used to take up plasmid DNA from the ligation reaction. An infected cell produces 100-200 double-stranded (or replicative form (RF) copies of the phage genome.

These encode genes that coordinate packaging of one of the phage genome DNA strands into a proteinaceous viral coat and their extrusion from the cell where they are ...