Lin-Fa Wang

Promoter switching during development and the termination site of the σ43 operon of Bacillus subtilis

SummarySequencing data indicated that the RNA polymerase (σ43) operon of Bacillus subtilis consisted of three genes, P23 (function unknown), dnaE (DNA primase), and rpoD (σ43) (Wang and Doi 1986a). S1 nuclease mapping experiments with RNA from various stages of growth demonstrated the presence of two overlapping σ43 promoters that controlled the expression of the operon during growth and a σ37 promoter that regulated the expression of the operon during the sporulation phase. This promoter switching mechanism ensured that this important operon would be expressed during different nutritional states of the cell and also illustrated a function for the minor RNA polymerase σ37 holoenzyme in the expression of genes which are normally expressed during the logarithmic phase of growth. The location of the transcription termination signal confirmed that the σ43 operon consists of three genes.

Expression and secretion of human atrial natriuretic α-factor in Bacillus subtilis using the subtilisin signal peptide

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter. The hANF is the first human gene product to be secreted from B. subtilis using this signal peptide. We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs. In addition, both cell growth and sporulation are adversely affected by hANF production. Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide.

Expression of a full-length nonstructural protein NS1 of bluetongue virus serotype 17 in Escherichia coli

The relative abundance of the nonstructural protein NS1 in bluetongue virus (BTV)-infected cells, the existence of NS1 in the BTV particles and the highly conserved NS1 gene among BTV serotypes indicate the diagnostic potential of using NS1 in detecting BTV infections. In this study a NS1 gene was expressed with the T7 RNA polymerase expression system to produce a full-length NS1 protein. Sheep anti-NS1 antibodies were raised with the E. coli-produced NS1 and used to show that the NS1 proteins of the five BTV serotypes in the Unites States were immunologically indistinguishable.

Bluetongue virus-17 fusion protein NS1 expressed in Escherichia coli by pUC vectors

The cDNA coding for the major nonstructural protein, NS1, of bluetongue serotype 17 (BTV-17) was cloned previously. Using pUC plasmids, we have successfully expressed the NS1 protein in Escherichia coli as a LacZ-NS1 fusion protein. The recombinant NS1 protein reacted with rabbit anti-BTV-17 antiserum, and was thus immunologically indistinguishable from the native BTV-17 NS1 protein. This was the first bluetongue viral protein to be produced in a bacterial system.