大肠杆菌、枯草杆菌穿梭表达载体的构建及改造

将大肠杆菌的复制子rep和多克隆位点克隆到枯草杆菌质粒pGDV1的骨架上,即得到大肠杆菌枯草杆菌牙梭庾粒载俸pGDVM。在pGDVM上进行载体表达元件的构建,先后将P59启动子、核糖体结合位点SD和终止子克隆到pGDVM上得到穿梭表达载体GJ01。以β-半乳糖苷酶基因（bga）作为报告基因检测载体的表达活性,在大肠杆菌和枯草杆菌中β-半乳糖苷酶（Bga）酶活性最高达到75.3和83．2个密勒单位,表明所构建的表达载体具有较强的表达能力。以核糖体结合位点（SD、SD3、SD4和SD5）代替表达载体GJ01-bga中的SD,对载体进行改造。所构建的GJD2-bga在大肠杆菌中的最大酶活性为253．8个密勒单位,G]D5-bga在枯草杆菌中的最大酶活性为135．4个密勒单位,表明所构建的载体具有较强的表达活性。由此可以得出不同的SD序列及其与起始密码子的距离不同程度地影响mRNA的翻译效率。     

Assay and characterization of a strong promoter element from B. subtilis

A new strong promoter fragment isolated from Bacillus subtilis was identified and characterized. Using the heat stable beta-galactosidase as reporter, the promoter fragment exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned and the beta-Gal directed by the promoters was high-level expressed both in E. coli and B. subtilis. By means of the isolated promoter, an efficient expression system was developed in B. subtilis and the benefit and usefulness was demonstrated through expression of three heterologous and homogenous proteins. Thus, we identified a newly strong promoter of B. subtilis and provided a robust expression system for genetic engineering of B. subtilis.     

Use of synthetic ribosome binding site for overproduction of the 5B protein of insertion sequence IS5.

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Although there was no detectable expression from the 5B open reading frame when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. beta-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.

The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E. coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.