PROTEINASE-K FROM TRITIRACHIUM-ALBUM-LIMBER - CHARACTERIZATION OF THE CHROMOSOMAL GENE AND EXPRESSION OF THE CDNA IN ESCHERICHIA-COLI
PROTEINASE-K FROM TRITIRACHIUM-ALBUM-LIMBER - CHARACTERIZATION OF THE CHROMOSOMAL GENE AND EXPRESSION OF THE CDNA IN ESCHERICHIA-COLI
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DOI：
10.1111/j.1432-1033.1989.tb14539.x
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发表时间：
1989-01-15
期刊：
影响因子：
--
通讯作者：
GASSEN, HG
中科院分区：
文献类型：
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作者：
GUNKEL, FA;GASSEN, HG
在大肠杆菌中克隆了小檗（Tritirachium album Limber）的cDNA和编码蛋白酶K的染色体基因，并确定了编码区以及5′和3′侧区的全部核苷酸序列。导出的初级翻译产物由384个氨基酸残基（分子质量= 40231 Da）组成，其中n端含有成熟蛋白中不存在的105个氨基酸。通过与进化相关的细菌枯草菌素和其他丝氨酸蛋白酶的类比，推断其主要分泌产物是含有15个氨基酸信号序列和90个氨基酸前肽的酶原。前肽可能在分泌过程的后期或在分泌到培养基中时被去除。该基因及其侧翼区域的核苷酸序列分析表明，蛋白酶- k基因由两个外显子和位于前区域的一个63 bp长的内含子组成。此外，一个假定的启动子序列和一个capping位点已经被确定，这表明转录起始位点位于ATP起始密码子上游103-bp处。为了在大肠杆菌中表达原蛋白酶- k基因，在tac启动子控制的质粒载体上克隆了原蛋白酶- k cDNA。为此目的构建的杂交质粒pSPPRO包含编码原蛋白酶K的cDNA[从Ala（. sdb .91）到c端Ala(279)]，与以tac启动子为先导的碱性磷酸酶基因的n端信号肽序列融合。携带这种质粒的大肠杆菌BMH71-18在脱脂牛奶培养皿上显示出轻微的蛋白水解活性，这表明一些融合蛋白被正确地分泌到外质中，并被加工成成熟的蛋白酶K。
The cDNA and the chromosomal gene encoding proteinase K from Tritirachium album Limber have been cloned in Escherichia coli and the entire nucleotide sequences of the coding region, was well as 5''- and 3''-flanking regions have been determined. The deduced primary translation product consisting of 384 amino acid residues (molecular mass = 40231 Da) contains an N-terminal region of 105 amino acids not present in the mature protein. By analogy to the evolutionary-related bacterial subtilisins and other serine proteinases it is inferred that the primary secreted product is a zymogen containing a 15-amino-acid signal sequence and a 90-amino-acid propeptide. The propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. The nucleotide-sequence analysis of the gene and its flanking regions has revealed that the proteinase-K gene is composed of two exons and one 63-bp-long intron located in the proregion. Furthermore, a putative promoter sequence and a capping site have been identified, suggesting that the transcription-start site is located 103-bp upstream of the ATP initiation codon. To express the proproteinase-K gene in E. coli, proproteinase-K cDNA was cloned in a plasmid vector under control of the tac promoter. The hybrid plasmid pSPPRO, constructed for this purpose contained the cDNA coding the proproteinase K [from Ala (.sbd.91) to the C-terminal Ala(279)] fused to the N-terminal-signal-peptide sequence of the alkaline-phosphatase gene preceded by the tac promoter. E. coli BMH71-18, harbouring this plasmid, exhibited slight proteolytic activity when tested on skimmed-milk plates, suggesting that some fusion proteins were correctly secreted into the periplasm and processed to the mature proteinase K.