Invitro Synthesis of Luciferase protein using rabbit reticulocyte cell expression system

Abstract


T7 RNA polymerase is a DNA dependant polymerase and can catalyze RNA synthesis in the

5′→3′ direction with the help of a promoter sequence. It can be used to produce proteins

efficiently in vitro transcription followed by in vitro translation processes. This study aimed

to express and purify T7 RNA polymerase and use it to produce the protein of interest

through in vitro assays. The T7 RNA polymerase was expressed by using BL21 cells induced

with IPTG. The T7 RNA polymerase was isolated by Ni NTA affinity chromatography. After

obtaining the T7 RNA polymerase, its purity was checked using SDS PAGE and Bradford

assays. The RNA polymerase was found to be pure and the obtained concentration was

consistent with the Bradford assays performed. At the same time plasmid that encodes for

luciferase was obtained by using DH5α cells. The plasmid DNA was linearized by the

digestion with Xbal. The linearized plasmid along with the purified T7 RNA polymerase was

used in in-vitro transcription to generate luciferase mRNA. The T7 RNA polymerase

obtained was found to be pure and effective as the mRNA transcript was produced

efficiently. There was some deviation from the results expected. As the in vitro translation

did not yield the luciferase protein. The Green fluorescence indicating the successful in vitro

translation was not observed in the experimental and negative control samples. However, the

positive control gave fluorescence. The reasons for this result were investigated along with

the possible solutions.

, Introduction


In vitro construction of biological systems can help in understanding many processes in

biology. In vitro translation and transcription can mimic living cells and synthesize proteins

from mRNA or DNA in test tubes. ( Fujiwara,2017). The in vitro transcription systems are

used to detect and study molecular mechanisms of transcription. In in vitro systems, the

reaction environment can be controlled directly as there is no cell barrier. For this process

DNA dependant, RNA polymerase such as T7 RNA polymerase is required along with a

number of transcription factors. In vitro transcription can help in understanding the complex

regulatory steps involved in transcription. A linear DNA fragment of DNA having a promoter

region is required as a template for this process. This linear DNA fragment can be generated

by linearizing a plasmid or by PCR . ( Yang & Ma, 2016). The T7 RNA polymerase is DNA

dependant RNA polymerases consisting of only one subunit. It is a 99 kDa RNA polymerase

derived from T7 bacteriophage. The T7 RNA polymerase efficiently transcribes the genes

bearing the T7 promoter. This polymerase is similar to RNA polymerase found in chloroplast

and mitochondria, DNA-directed DNA polymerases, and reverse transcriptases .T7 RNA

polymerase is highly specific and transcribes products with high incorporation fidelity. It is

able to transcribe a gene completely without using any additional proteins. (Borkotoky &

Murali, 2018).


T7 RNA polymerase can synthesize RNA from double as well as single-stranded RNA in the

presence of a double-stranded DNA promoter sequence. Also, T7 RNA polymerase can also

produce RNA without the promoter DNA.( Gholamalipour,2018). The RNA polymerases can

be purified by the affinity chromatography technique. Affinity chromatography is a liquid

chromatographic system used for the purification, separation, and analysis of samples.

Affinity chromatography acts by binding protein to a ligand in a matrix. In the affinity

chromatography technique, the stationary phase is made of a support medium where the