Dahl S W, Slaughter C, Lauritzen C, Bateman R C, Connerton I, Pedersen J
Unizyme Laboratories A/S, Dr. Neergaards Vej 17, Horsholm, Denmark,
Protein Expr Purif. 2000 Oct;20(1):27-36. doi: 10.1006/prep.2000.1273.
A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.
基于对天然番木瓜谷氨酰胺环化转移酶胰蛋白酶片段的氨基酸测序结果，通过逆转录聚合酶链反应（RT-PCR）克隆了编码该酶的全长cDNA。1036个核苷酸的cDNA编码一个典型的22个氨基酸残基的信号肽和一个266个氨基酸残基的成熟蛋白，计算分子量为30923道尔顿。鉴定出5个编码与番木瓜谷氨酰胺环化转移酶高度同源的假定谷氨酰胺环化酶的植物EST，半胱氨酸和N-糖基化位点的数量和位置是保守的。植物谷氨酰胺环化酶的氨基酸序列与已知的哺乳动物谷氨酰胺环化酶序列非常不同，未观察到明显的同源性。番木瓜谷氨酰胺环化转移酶cDNA在大肠杆菌中表达为带有三种不同信号肽的His标签番木瓜谷氨酰胺环化转移酶，以及与硫氧还蛋白、谷胱甘肽S-转移酶和（前）麦芽糖结合蛋白的融合蛋白。在所有情况下，表达的蛋白要么无法检测到，要么不溶。与α-因子前导肽融合的番木瓜谷氨酰胺环化转移酶在毕赤酵母中的表达导致低水平的番木瓜谷氨酰胺环化转移酶活性。番木瓜谷氨酰胺环化转移酶在昆虫细胞/杆状病毒系统中的细胞外表达成功，表达并纯化了15-50毫克/升带有三种不同分泌信号的活性番木瓜谷氨酰胺环化转移酶。此外，N端与分泌信号/His标签肽组合融合的番木瓜谷氨酰胺环化转移酶被宿主信号肽酶正确加工，随后His标签可用二肽基肽酶I去除。通过活性测定、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、N端氨基酸测序、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和肽质量指纹分析对表达产物进行了表征。
