Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Abstract

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant

“…The low stability of the CP subunits may also explain the puzzling fact of an absence of the assembly activity of TMV CP molecules produced in Escherichia coli cells [24,25]. Probably, newly synthesized CP molecules can be correctly folded only with the help of some specific chaperones not present in E. coli cells.…”

Aromatic and Cystine Side-Chain Circular Dichroism in Proteins

“…Near-and/or far-UV CD have been used frequently in combination with denaturing conditions such as elevated temperature, low or high pH, or urea or guanidine to assess the stability of the mutant or modified protein relative to that of the wild-type protein (Elwell and Schellman, 1977;Wendt et at., 1988;Shire et at., 1990;Brems et at., 1990;Wingfield et at., 1991;Zhang et at., 1993;Khorasanizadeh et at., 1993;Yang et al, 1994).…”

Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

“…The resulting particles were shorter than with plant-derived TMV CP and had a striated or stacked cylindrical appearance. More recently, Shire et al (22) expressed TMV U1 CP in E. coli from a trp promoter. CP was purified from cleared cell lysates by chromatography and dialysis (precipitation) at pH 3.5.…”

“…The N-terminal N-formyl-Met had been removed, but the penultimate N-terminal Ser was not acetylated, unlike plant-encoded TMV CP. This feature, or the added positive charge, disrupted formation of helical CP aggregates and the 20S assembly nucleation species (7)(8)(9)(10) and created assembly-incompetent CP when incubated with TMV RNA in vitro (22,23). In our study, two point mutants of the U1 CP sequence were created to change the mature N terminus to Ala or Pro, which exist without acetylation in the NM and U2 strains of TMV, respectively (24).…”