Modified CNBr Cleavage Protocol for Efficient Separation of Met-Ser Containing OmpX-Om14 Membrane Protein Fusion

Ankit Gupta(1*), Deepti Chaturvedi(2), Radhakrishnan Mahalakshmi(3)

(1) Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Transit campus : ITI (Gas Rahat) Building, Govindpura, Bhopal 462023. India., India
(2) Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Transit campus : ITI (Gas Rahat) Building, Govindpura, Bhopal 462023. India., India
(3) Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Transit campus : ITI (Gas Rahat) Building, Govindpura, Bhopal 462023. India., India
(*) Corresponding author


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Abstract


Cleavage efficiencies of cyanogen bromide (CNBr) are drastically reduced in sequences wherein the methionine residue is succeeded by serine or threonine. Solubility issues associated especially with membrane proteins contribute further to reduced cleavage at Met-Xxx sites. Moreover, prolonged incubations of several hours to days, in highly acidic reaction conditions required for CNBr cleavage, often lead to protein degradation. To circumvent these problems, we have developed a protocol for CNBr cleavage of protein fusions, which is particularly useful for Met-Ser sequences. This protocol facilitates enhanced cleavage efficiencies in short incubation times of 1-2 hours. It incorporates up to 40% acetonitrile in 8M urea, or trace amounts of acetonitrile in 6M guanidine hydrochloride, in the final reaction. We demonstrate the successful application of this protocol for the Met-Ser containing membrane protein fusion OmpX-Om14. Cleavage of the Om14 (the yeast mitochondrial outer membrane protein) from E. coli OmpX (Outer membrane protein X) has provided yields of up to 70% product without detectable degradation levels. The direct use of the cleavage reaction for the rapid and cost-effective purification of Om14 has been shown. Using a database analysis, we discuss the importance of this protocol for several membrane proteins that have Met-Ser sequences
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Keywords


Met-Ser sequence; CNBr Cleavage Efficiency; Membrane Proteins; Fusion Construct; Acetonitrile; Insoluble Protein; Inclusion Bodies

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