Expression of tRNase Z using BEV (Lab Report Sample)

Expression of tRNase Z using BEV
Bio 301 Molecular Biology and Biotechnology Lab 4
Professor:
December , 2021
Abstract
This lab is aimed at cloning a tRNase Z gene using a Bacidiovirus Expression Vector (BEV), allow for the protein expression and further use the information to determine the amount of virus that can bring forth an optimum protein yield. In order to achieve this, a cDNA of the gene of interest is made. Addition of reverse transcriptase enzyme to a purified mRNA sample aids the synthesis of the first copy of the cDNA using the RNA strand as a template. The DNA polymerase enzyme then synthesizes the second copy of the cDNA resulting to an intron-less double stranded DNA. The cDNA strand produced is then cloned into the BEV. Once the cloning is done, the recombinant BEV is then used to transfect the Sf9 cell cultures. In this experiment, it was realized that for the tRNase ZS, 5 μl of bacidiovirus infected cells gave the highest yield of the recombinant protein while tRNase ZL on the other hand took 15 μl to produce the highest protein concentration.
Introduction
Baculovirus Expression Vector (BEV) System
Baculovirus belongs to class of DNA of viruses that exhibits a large covalently-well closed double stranded DNA of approximately 88-200kbp, which is highly endowed with basic proteins (Luckow & Summers, 1989) encapsulated in a nucleoplasmid containing capsid proteins. The packaging of the nucleoplasmid in a lipoprotein forms a virion which may be covered in a polyhedron made up of 30kDa single protein polyhedrin (Luckow & Summers, 1989). The Baculivirus has two forms of the virus; the ECV and the polyhedral. The virus particles infects cells through adsorptive endocytosis, and moves to the nucleus for its replication. The presence of a gene promoter in this virus can be harnessed in the development of the BEV system, using Autographa caliifornica nuclear polyhedrosis virus which has a double stranded DNA.
The cells that are able to undergo successful recombination can be separated from those that does not, through the absence of the polyhedrin, on the infected cells when visualized under a microscope. The polyhedrin functions as a protective the external environmental conditions in the wild type Baculovirus.
Histidine- tagged Affinity Chromatography
The purification of the protein (tRNaseZ) is done through Histidine –tagged affinity chromatography. This method employs a technique of purification called Immobilized Metal Affinity Chromatography (IMAC) ("Purification of his-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent," n.d.). Here, there is an immobilization of transition metal ions on a matrix of resin by the use of chelating agents. Nickel ion is used here in histone-tagged purification, where there is high affinity of histidine-tag to Nickel ions, and they tightly bind to the IMAC column. The viral tRNase Z protein do not bind to the resin, or if they do, it happens weakly. For the elution of the pure form of the proteins on the IMAC column, Imidazole is used. Imidazole competes for affinity with the histidine-tag to the charged metal resin (Zachariou, 2008). The imidazole, is added in low concentrations to both wash and binding buffer to tamper with other proteins that binds weakly and to elute these weakly binding proteins. Nickel ions are therefore preferred because they give a high yield.
Complementary DNA (cDNA)
The cDNA are made through a reverse transcription process from purified mRNA units. The purification of the mRNA takes place in an OligodT cellulose column where its polyA tail hybridizes with the attached poly T primers (Clark & Pazdernik, 2013). Addition of the Reverse transcriptase enzyme to the bound messenger RNA creates the first copy of the cDNA in a 5’-3’ direction. The DNA polymerase enzyme synthesizes the second strand copy of the cDNA using the first copy as a template. This results to double stranded cDNA with no introns. The RNase hybridase cuts and breaks apart RNA in the RNA in cDNA – RNA hybrid. The resulting cDNA can be used as a gene of interest insert, as it contain an Open Reading Frame (ORF) (Clark & Pazdernik, 2013).
Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The reaction between acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide) results in the formation of polyacrylamide gel, a cross-linked gel matrix (Magdeldin, 2012). This gel has sieve-like structures whereby the proteins move relative to electric field applied. The proteins to be separated must be pre-treated with SDS to generate a uniform negative charge, heating up to temperatures of 95 - 1000C to enature the protein structures (Boldura & Baltă, 2018). Mercaptoethanol or Dithiothreitol (DDT) are further added to break all the covalent disulfide bonds in the peptides. The resulting residue can therefore be separated based on their apparent molecular weights influenced by their relative mobilities.
Methodology
Pre-lab preparations.
Invitrogen pFastBac™HT plasmid expression cloning vector. First, the gene of interests’ cDNA is cloned into the MCS (multiple cloning site) of the expression vector, the recombinant plasmids are then utilized to transform DH10Bac, a special E. coli cells strain construct that are transformation-competent. The DH10Bac strain has both complete AcMNPV viral genome in the plasmid, as well as an E.coli Transposon 7 (Tn7) mini-attTn7, a transposition target site downstream of the gene promoter of the Polyhedrin, and a helper plasmid encoding for the essential transposition proteins that transposes the Tn7R through Tn7L as shown in the map. This is done inside pFastBac™HT A into the Bacmid plasmid target. After the transposition reaction, high molecular weight recombinant Bacmid was then isolated and used to transfect the Sf9 insects’ cells thereby generating the recombinant baculovirus used in this lab which were frozen and stored. The pFastBac™HT has the following structure.
Lab Session week 11
Ultra-cold frozen pellets of the recombinant baculovirus-infected Sf9 insect cells were obtained from the CLT and thawed by holding them in the warm fingers of a gloved hands till they become slushy. The cells were then resuspended in a lysis buffer containing 0.5% NP-40 using a micropipette, followed by micropipete sucking and release for five times. Followed by shaking 15 minutes in a cold room temperature to disrupt the cells plasma membrane and the flasks labelled with the group name and flask number.
The flask was gently swirled in order to ensure a uniform cell suspension, then transferred by pouring it into a pre-labeled Falcon capped centrifuge tube. The capped tube was subjected into a low-speed controlled-temperature centrifugation at 500 RPM for 5 min.
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As the tubes are still rocking, a Qiagen Ni-NTA spin column was labelled. Equilibration of the Ni-NTA spin column was done with 600 μl Lysis Buffer, followed by centrifugation of the column at 890 x g for 2 min.
Once the cold incubation for lysis of the Sf9 cells was complete, each tube of cell lysate was elucidated by centrifuging for 15 min at 12,000 x g in a refrigerated microcentrifuge and the clear cl lysate on top of the pellet collected. 2 μl of the cleared lysate (CL) was transferred into a tube having 20 μl of protein loading buffer (PLB) and then kept on ice.
The main CL volume was loaded onto a pre-equilibrated Ni-NTA spin column then centrifuged for 5 minutes at 270 x g in a microcentrifuge in the cold room, the resulting the flow-through (FT) solution was poured from the collection tube back into the column and spin. 2 μl of the flow-through was transferred into 20 μl tube containing protein loading buffer to check the efficiency of the binding of the 6XHis-tagged protein in the spin column procedure and kept in a cold lab condition for the next lab.
The Ni-NTA spin column was washed twice with a 600 μl of Wash Buffer, after every wash, centrifugation for 2 minutes at 890 x g was done at room temperature and the washes discarded.
300 μl Elution Buffer was added into the column to elute the proteins and centrifuged for 2 minutes at 890 x g and EL1 solution collected. A repeated with an additional 200 μl of Elution Buffer and EL2 solution collected. The total eluted volume was saved and marked EL as the main eluted material for analysis in SDS-PAGE.
2 μl of the EL was transferred into a tube containing 20 μl of PLB to check for efficiency of expression and affinity purification of the His-tagged protein then frozen in ice.
The fractions of the samples in PLB buffer that were kept on ice were retrieved, gently mixed and placed in a boiling water bath for 90 seconds followed by centrifugation for 230 seconds and the resulting liquids numbered as shown.
Tube # (G=Group) (n=member #)

Fraction

G# 1-CL

Cleared Lysate

G# 2-FT

Flow-through

G# 3-EL

Eluate

left22053556XHis Tag and Affinity Purification006XHis Tag and Affinity Purification
The CL and FT of the columns were saved for later use in the tracking of the affinity purification progress.
The buffers used were prepared using the ingredients and their respective concentrations, volumes and pH conditions showed below.
Lysis Buffer (100 ml)
IngredientsStockFinal []Volume
Na-PO4 (pH 7....