• Project Description
• Overview
• Background

• Ag43 Secretion System
• Overview
• Design
• Experiments
• Result
• Conclusion

• Other Systems
• His-tag Inactivation System
• Co-expression System
• Discussion

• Future Work

• Modeling

His-tag Inactivation System

Overview

One of the ways to produce large quantities of a recombinant toxic protein in E.coli is to aggregate it as inclusion bodies (IBs). IBs are insoluble protein aggregations observed in bacteria overexpressing heterologous genes whose products fail to attain a soluble (de Groot et al. 2008). Protein aggregation can lose the activity of the protein to some extent. So we optimized inclusion bodies to achieve large amounts of the toxic peptide, thanatin.

It’s known that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies. In our project, we intended to express thanatin with a 10x His-tag at the N terminus as an inclusion body, then cleave of a His-tag with some protease (TEV protease) leading successfully to express thanatin in a soluble form.

• Inclusion bodies (IBs)

Inclusion bodies (IBs) are major protein aggregates commonly occurring in recombinant bacteria when the expression of plasmid-encoded genes is directed at high rates

• His tag

His tag (histidine-tag) is an amino acid motif consisting of successive histidine residues, often at the N- or C-terminus of the target protein. His tag is often used for affinity purification of tagged recombinant proteins expressed in Escherichia coli.

• TEV potease

TEV protease is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). TEV protease uses a cysteine as its catalytic nucleophile. The native cut sites of this protease is Glu-Asn-Leu-Tyr-Phe-Gln-/-(Gly/Ser) where ‘/’ is the cleaved peptide bond

Design

First, we made the construct like figure 1 (Plac-B003x-Thanatin-dT on 1C3). Thanatin is under control of one of inducible promoters; Plac promoter. In the absence of IPTG, lacI protein act as repressor and bind to promoter region, therefore expression of downstream gene of Plac is negatively controlled. By adding IPTG, this inducer interact with lacI and cause the conformation change of repressor. Finally, lacI is released from promoter region, leading recruiting of RNA polymerase and initiation of transcription. We predict that E.coli could be difficult to grow due to leak of Plac (Even if we don’t induce by IPTG downstream gene would be transcribed and thanatin could kill host cells)

Second, we made the construct like figure 2 (Plac-B003x-10xHistag-Thanatin-dT on 1C3).We designed TEV cleave site between 10xHistag and Thanatin.

Experiments

We try to reserch 10xHis tag fusion with thanatin and formation of inclusion body can surely keep thanatin inactive and TEV protease can cleave His tag and get thanatin to be reactive.

How assay

First of all, we make sure that the constructs have correct sequence by sequencing.If we get the expected constructs, we transform it into JM109 and compare the growth curve among different inductive condition.

1. Activity test for Thanatin and 10xHIS thanatin
→Comparing growth curve between the samples one is induced by IPTG and the other is not induced. Diluting the bacterial culture which is enough pre-cultured by 0.1 O.D.600 (Optical Density in λ=600nm) in 2ml LBC.(Lysogeny Broth liquid culture including chloramphenicol)
2. Adding 2µl of IPTG into one and DW into the other.
3. Measuring O.D.600 of these samples once in an hour.

Reactivation of thanatin by TEV proteinase

1. Giving damage to the bacteria body by ultrasonic treatment.
2. Adding TEV protease and buffer to the homogenate
3. Centrifuge 5,000rpm for 2min
4. Conducting MIC test for the supernatant

Result

Conclusion

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Co-expression System→