Szweda Piotr, Kotłowski Roman, Kur Józef
Department of Food Chemistry, Technology and Biotechnology, Gdańsk University of Technology, ul. G. Narutowicza 11/12, 80-952 Gdańsk, Poland.
J Biotechnol. 2005 May 4;117(2):203-13. doi: 10.1016/j.jbiotec.2005.01.012.
The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.
pET23b+（纽英伦公司，英国）和pBAD/Thio-TOPO（英杰公司，美国），这两种系统可使目标蛋白作为融合蛋白过量表达。在pET系统中产生的酶在C末端含有一组六个组氨酸，而在pBAD系统中产生的蛋白在N末端含有另外133个氨基酸残基，包括硫氧还蛋白、一组六个组氨酸和一种内蛋白酶Xa因子的识别位点。重组酶通过在Co2+-琼脂糖柱上进行金属亲和层析来纯化。从1升培养物中，在pET表达系统中获得了约20毫克纯化的重组酶，在pBAD系统中获得了39毫克。从pET系统获得的融合蛋白显示出的比活性比从pBAD系统获得的融合蛋白高约10倍（分别为970 U/mg和83 U/mg）。纯化后的酶分别在接近45摄氏度和pH 8.0（对于从pET系统获得的酶）或pH 7.5（对于从pBAD系统获得的酶）时表现出最大活性。Zn2+或Cu2+（2 mM）强烈抑制溶葡萄球菌酶的活性，活性降低70 - 80%。Ni2+（2 mM）也抑制该酶，对于从pET和pBAD系统获得的酶，活性分别降低60%和20%。2 mM浓度的Co2+对酶活性没有影响；然而，在10 mM浓度时，对于从pET和pBAD表达系统获得的酶，活性分别降低30%和20%。已知作为天然溶葡萄球菌酶的强抑制剂的EDTA，对两种重组酶的抗葡萄球菌活性均无影响。
