High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris.
Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.
Jin
Feng-liang
FL
College of Natural Resources and Environments, South China Agricultural University, Engineering Research Centre of Biological Control, Ministry of Education, Guangzhou 510642, China.
Xu
Xiao-xia
XX
Yu
Xiao-qiang
XQ
Ren
Shun-xiang
SX
D000818:Animals; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015870:Gene Expression; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D043222:Ubiquitin Thiolesterase