Peptide mapping/Western immunoblot procedure for identification of a truncated mycoplasma protein expressed in Escherichia coli

https://doi.org/10.1016/0167-7012(91)90017-KGet rights and content

Abstract

The analysis of mycoplasma genes cloned into Escherichia coli is often difficult because gene products, if expressed at all, can be truncated due to unusual codon usage. Identification of cloned proteins can be further complicated in the absence of a known biological activity for which to screen. By combining two-dimensional sodium dodecylsulfate-polycrylamide gel electrophoresis, in situ proteolysis, and immunoblot analysis, we have developed a technique for analyzing clone proteins based upon their peptide profiles. While this concern is not new, our approach allows analysis directly by sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western immunoblotting, without prior purification from comigrating proteins. Mycoplasma pneumoniae proteins and proteins from an E. coli clone expressing most of the mycoplasma protein HMW3 (HMW3) were compared following Staphylococcus aureus protease V8 digestion, yielding identical profiles of a single low molecular weight spot. Digestion with chymotrypsin gave similar profiles for clone and wild-type cells, with six common peptides identified. Minor differences observed in the peptide patterns can be expected when comparing a unit-length protein with its truncated homolog.

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