Chemical degradation of fusion proteins

Summary

The use of gene fusion expression systems to express exogenous genes in E. coli has become increasingly popular. The reason for this is largely attributed to the ability of fusion systems to produce large amounts of soluble fusion proteins. Glutathione transferase as well as thioredoxin have been shown to be very successful in producing correctly folded, biologically active proteins. Each of these has a convenient purification method to separate the fusion protein from cellular contaminants. The resulting proteins are suitable for conducting studies of their biological activity or (and) interactions. As fusion expression methods become more common, the ability to cleave the N-terminal portion of the carrier protein from the C-terminal target protein becomes more important. Cyanogen bromide (CNBr) has been used in protein cleavage reactions for many years, typically in 70% formic acid at very low pH, with cleavage occurring at the C-terminus of methionine. Sourced from Compact Molecular Biology Laboratory Guide (5th Edition)

Operation method

Basic program Cyanogen bromide

Materials and Instruments

1 mg ml fusion protein
50 mg ml CNBr 70% (V V) formic acid 1 × SDS sample buffer

Move

1. Perform a pre-test to determine the minimum incubation reaction time. Lyophilize two small portions of 50 μl each of the fusion protein solution, resuspend one portion in 50 μl of 50 mg/ml CNBr/70% formic acid, and resuspend the other portion in 50 μl of 70% formic acid without CNBr, and allow the reaction to take place at room temperature. 2.

2. At 0, 8, 24 and 48 h, take 5 μl of each sample and lyophilize. 3.

3. Add 20 μl of 1 × SDS sample buffer to all the lyophilized samples, boil for 10 min, and add the samples to an SDS-polyacrylamide gel. 4.

4. Analyze the gel electrophoresis results to determine the minimum incubation reaction time required for complete cleavage of the protein.

Caveat

1. CNBr is highly toxic and should be handled in a fume hood and used and disposed of with care.

2. When the protein is resistant to CNBr cleavage or when the protein to be cleaved is an insoluble fusion protein, guanidine hydrochloride may be added to the reaction to a final concentration of 6 mol/L.

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