A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies.

Derivatization with trimethylammoniopropyl methanethiosulphonate (TAPS-sulphonate) enabled brain-derived neurotrophic factor (BDNF) to be prepared efficiently from Escherichia coli inclusion bodies. Reduced BDNF obtained from inclusion bodies solubilized by urea and reduced by dithiothreitol was suggested to form a complex with itself or with other compounds such as lipids. It could hardly be adsorbed on to cation-exchange resin for partial purification prior to a refolding reaction. Reversible derivatization of cysteine residues was tested as a method of dissociating BDNF from such complexes. However, even if a methyl or aminoethyl group was introduced, BDNF could not be dissociated readily. Derivatization with TAPS-sulphonate brought about good dissociation of BDNF, and more than 50% adsorbed on to the cation-exchange resin. BDNF derivatized with TAPS-sulphonate refolded well, and the refolded samples showed the same biological activity as purified BDNF. Derivatization with TAPS-sulphonate would increase the intermolecular repulsion of BDNF, due to the positively charged character of the quaternized amine, and inhibit complex formation. Thus, TAPS-sulphonate is effective for the preparation of BDNF under denatured conditions.

Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli.

Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.

SecY is an indispensable component of the protein secretory machinery of Escherichia coli.

Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.

Purification of SecE and reconstitution of SecE-dependent protein translocation activity.

SecE was solubilized from SecE-overproducing E. coli cells and purified through ion exchange and size exclusion chromatographies. When the solubilized membrane containing overproduced amounts of SecY and SecE was fractionated by means of size exclusion chromatography, the two proteins were eluted in different fractions with slight overlapping. Proteoliposomes active in protein translocation were reconstituted from these fractions only when both SecE and SecY were present. When reconstitution was carried out with the purified SecE and fractions containing SecY but only a small amount of SecE, the resultant proteoliposomes exhibited appreciable translocation activity, indicating that SecE is essential for protein translocation. The translocation activity of proteoliposomes was proportional to the amount of purified SecE used for reconstitution. SecE-dependent protein translocation absolutely required ATP and SecA.

SecE-dependent overproduction of SecY in Escherichia coli. Evidence for interaction between two components of the secretory machinery.

The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids. Induction with isopropyl-beta-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY. The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE. SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were. It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane. The numbers of the SecY and SecE molecules per cell were estimated.

An insertion mutation associated with constitutive expression of repressible acid phosphatase in Saccharomyces cerevisiae.

The PHO83 mutation in Saccharomyces cerevisiae, which had been detected on the basis of constitutive production of repressible acid phosphatase and mapped at the end of the PHO5 locus, was analysed by Southern hybridization with cloned DNA fragments of the PHO5 gene as probe. It was shown that this mutant has a DNA insertion of about 6 kilobase pairs, probably in the 5'-noncoding region of the PHO5 gene. Production of repressible acid phosphatase by the PHO83 mutant is partially independent of the function of the PHO2 and PHO4 genes, the positive regulatory genes whose functions are indispensable for PHO5 expression. PHO83 mutants are constitutive in a and alpha cells, either haploid or diploid, but not in non-mating cells, MATa/MAT alpha or a certain sterile mutation. These observations strongly suggest that the PHO83 mutation is caused by insertion of a Ty element in the 5'-noncoding region of the PHO5 gene.