Expression and Purification of Highly Toxic Barnase Protein in Bacterial System

FEATURED CASE STUDY

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This case study describes a process for the expression and purification of highly toxic Barnase protein from barnase-barstar co-expression construct for acute toxicity studies using E. coli host system.

Introduction:

Magnitude of heterosis is much lower in self-pollinated crops than cross-pollinated crops. Therefore, improvement of self-pollinated crops by producing hybrid varieties are necessary and is a major goal of plant breeding. Several biotechnological strategies have been deployed to restrict self–fertilization in plants. However, the only commercialized transgenic male sterility method is SeedLink^TM, which relies on the expression of bacterial cytotoxic ribonuclease (Barnase) in the male reproductive organ of the female parent line and fertility restoration by ribonuclease inhibitor (Barstar) delivered by the male parent. However, issues such as leaky expression of the barnase gene and biosafety concerns associated with the use of the bacterial cytotoxic gene in food crops are the key challenges associated with its applicability. This explains the significance for allergenicity and acute animal toxicity studies on the Barnase protein, for regulatory approvals before its utilization in hybrid seed production.

Problem/Challenge:

Expression of Barnase in the cytoplasm is toxic to the E. coli host cell when a corresponding Barstar inhibitor is not co-expressed. This emphasizes the significance of expressing both barstar and barnase gene on a single plasmid for the production of active barnase in Escherichia coli. However, co-expression and purification of toxic Barnase protein from co-expression construct was a challenge for the client due to the simultaneous entry of Barstar and Barnase into the periplasmic space. Additionally, addition of any tag or extra amino acid at N or C terminus was leading to loss of activity of the protein. Therefore, objective of this study was to carry out monomeric expression of cytotoxic Barnase protein (without any tag or extra amino acids at N and C terminus) for acute toxicity studies; and its purification to produce >90% pure, highly active toxic Barnase protein.

Solution/Approach:

Premas Biotech has developed a potentially efficient co-expression and purification strategy to produce a lethal recombinant Barnase protein by using suitable host strain of E. coli, expression optimization and purification techniques. Optimization of expression conditions for Barnase was performed with two different host strains at different parameters such as the choice of host strain, induction temperature, and media formulations with various carbon sources, for high expression of functionally active protein.

Briefly, pET30-Barstar-Barnase co-expression construct was developed using sequential cloning procedures with insertion of pelB signal sequence to target Barnase to the periplasmic area. It was interesting to observe that despite the insertion of a pelB sequence, high levels of Barstar (which did not have a pelB sequence) were observed in the periplasmic space along with Barnase. This made purification of Barnase a more challenging task.

Purification Strategy:

Barnase-Barstar form a complex oligomeric structure stabilized by strong salt bridges and hydrogen bonding. A two-step purification strategy was developed to separate these proteins, while using urea denaturation to break the strong oligomeric interaction. Barnase, with a pI of 9.2 displayed poor binding to the cation exchanger, in presence of high amounts of Barstar. Barstar has a pI of 4.6; therefore, it was removed from the Barnase fraction using DEAE anion exchanger at a physiological pH (7.4). Binding affinity of Barnase to SP-FF and other resins were investigated using pH scouting. Optimum binding of Barnase with the SP-FF cation exchanger was observed at pH 4.5. Therefore, the Barnase protein was purified using SP-FF resin, and then refolding of the protein was performed by urea renaturation study using snap dilution method. The procedure resulted in a >90% pure, refolded Barnase Protein.

Fig.1. Purification of Barnase protein

Activity Analysis:

A non-radioactive, fluoremeteric assay was developed using Ethidium Bromide dye, to assess the RNase activity of the Barnase protein on yeast tRNA. Reaction was performed at constant amount of tRNA and varying concentrations of Barnase.

A continuous reduction in fluorescence implies RNA degradation by Barnase. The graph below states that the concentration of Barnase is directly proportional to the amount of yeast tRNA (RFU) degraded by the enzyme.

Fig.2. Activity analysis of Barnase

Mass-spectroscopic analysis further confirmed the expression of Barnase protein at the desired size.

Fig.3. Characterization of Barnase (by MALDI)

Measurable Impact:

The challenges encountered during the production of Barnase protein, have been resolved at Premas by developing a co-expression strategy with its inhibitor Barstar and then its purification to obtain monomeric Barnase. This process resulted in the production of high yields of a toxic protein, i.e. ~10mg/L of >90% pure recombinant Barnase protein. The Barnase protein was subsequently tested for allergenecity and acute animal toxicity studies, which was successfully completed and approved.

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