Battistoni A, Mazzetti A P, Petruzzelli R, Muramatsu M, Federici G, Ricci G, Lo Bello M
Department of Biology, University of Rome Tor Vergata, Italy.
Protein Expr Purif. 1995 Oct;6(5):579-87. doi: 10.1006/prep.1995.1076.
An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaperonins GroEL and GroES, a fraction of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may be obtained by genetic engineering technology.
构建了一种在大肠杆菌细胞质中大量产生谷胱甘肽 S-转移酶 P1-1（GST P1-1）的表达载体。纯化后获得的重组酶，对其理化性质和动力学性质进行了表征，结果显示其与从人胎盘中纯化得到的酶并无差异。然而，N 端氨基酸测序表明，约 50%的重组 GST 的 N 端氨基酸仍为甲硫氨酸。这种不完全加工并非仅仅是由于 GST 过量产生所致。事实上，在导致该蛋白产量急剧下降的生长条件下，N 端甲硫氨酸并未被去除。当 GST 与胡萝卜软腐欧文氏菌 pelB 基因的前导肽融合时，它无法穿过细菌膜，并以可溶且有活性的构象积累在细胞质中。然而，当在过量表达细菌伴侣蛋白 GroEL 和 GroES 的细菌菌株中产生这种融合蛋白时，一部分 GST 会以正确的 N 端序列输出到周质空间。正确加工的 GST 的产量占大肠杆菌细胞中总 GST 的 12%。我们的结果表明，伴侣蛋白能够与新生的 GST 相互作用，从而使该蛋白保持可输出的形式，并且通过基因工程技术有可能获得具有增强分泌特性的大肠杆菌菌株。
