Bingle W H, Smit J
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Plasmid. 1990 Sep;24(2):143-8. doi: 10.1016/0147-619x(90)90016-6.
A number of plasmid vectors were constructed for high-level gene expression in the dimorphic gram-negative bacterium Caulobacter crescentus. These vectors incorporate the transcription and translation initiation regions of the C. crescentus CB15A rsaA gene, which codes for the abundantly synthesized protein comprising the bacterium's paracrystalline surface layer. The expression vectors are based on the broad-host-range IncQ plasmid RSF1010 (R300B) and incorporate the rsaA promoter and transcription start site. Some vectors also contain translation initiation information; these can result in the addition of as little as a single glycine residue to the protein encoded by the cloned segment. The vectors can be introduced into C. crescentus by electroporation at high frequency (ranging up to 10(6)-10(7) electroporants/micrograms DNA with surface-layer-deficient mutant C. crescentus CB2A) or conjugal transfer. They range in size from 10 to 12 kb, specify either chloramphenicol or kanamycin resistance, and possess the restriction sites EcoRI, BamHI, KpnI, and SstI for cloning genes downstream of the rsaA gene sequences. For a number of the vectors, the complete nucleotide sequence is known. A comparison was made between the expression of an endoglucanase gene from these plasmids in C. crescentus CB2A and CB15A and similar constructions under the control of lacZ alpha transcription and translation initiation signals carried on a pUC9 vector in an Escherichia coli host. The two expression systems compared favorably; cell lysates prepared from C. crescentus CB2A exhibited 40% of the endoglucanase activity of similarly prepared lysates from E. coli JM101. Lysates prepared from C. crescentus CB15A exhibited only 8% of the endoglucanase activity of E. coli lysates.
构建了多种质粒载体，用于在革兰氏阴性双态细菌新月柄杆菌中进行高水平基因表达。这些载体整合了新月柄杆菌CB15A rsaA基因的转录和翻译起始区域，该基因编码构成细菌准晶体表层的大量合成蛋白。表达载体基于广宿主范围的IncQ质粒RSF1010（R300B），并整合了rsaA启动子和转录起始位点。一些载体还包含翻译起始信息；这些信息可导致在克隆片段编码的蛋白质上仅添加一个甘氨酸残基。这些载体可通过电穿孔（使用表层缺陷型突变体新月柄杆菌CB2A时，电穿孔效率高达10⁶ - 10⁷ 个电穿孔子/微克DNA）或接合转移高频导入新月柄杆菌。它们的大小在10至12 kb之间，赋予氯霉素或卡那霉素抗性，并具有EcoRI、BamHI、KpnI和SstI限制酶切位点，用于在rsaA基因序列下游克隆基因。对于许多载体，其完整的核苷酸序列是已知的。比较了这些质粒中的内切葡聚糖酶基因在新月柄杆菌CB2A和CB15A中的表达情况，以及在大肠杆菌宿主中由pUC9载体携带的lacZα转录和翻译起始信号控制下的类似构建体。这两种表达系统表现良好；从新月柄杆菌CB2A制备的细胞裂解物显示出大肠杆菌JM101类似制备的裂解物内切葡聚糖酶活性的40%。从新月柄杆菌CB15A制备的裂解物仅显示出大肠杆菌裂解物内切葡聚糖酶活性的8%。
