PRODUCTION, PURIFICATION, AND CLEAVAGE OF TANDEM REPEATS OF RECOMBINANT PEPTIDES
PRODUCTION, PURIFICATION, AND CLEAVAGE OF TANDEM REPEATS OF RECOMBINANT PEPTIDES
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DOI：
10.1021/ja00090a008
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发表时间：
1994-06-01
影响因子：
15
通讯作者：
WALSH, CT
中科院分区：
文献类型：
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作者：
KULIOPULOS, A;WALSH, CT
我们描述了一种构建多肽编码序列的串联重复序列的方法，所述多肽编码序列由单个甲硫氨酸残基散布并终止于His（6）标签，所述His（6）标签可以通过Ni螯合层析纯化，然后通过溴化氰（CNBr）切割成均质肽单元，编码13个氨基酸残基的酵母α-交配因子（α F）的互补42聚体寡核苷酸的退火和单向连接然后将寡聚的42聚体连接到pET载体中，该载体将α-因子串联重复序列置于酮类类固醇异构酶（KSI）基因的下游和His（6）盒的上游。在大肠杆菌中过量产生KSI-（alpha F）（5）-His（6）融合物，通过Ni螯合层析纯化，然后用CNBr裂解以释放不溶性KSI、His（6）尾和α-因子肽单元，每个单元均以高丝氨酸（HS）内酯终止。HPLC以56 mg/L的产率产生纯肽。α-因子-HS（内酯）可经氨解或水解分别产生α-因子-HS-酰胺或α-因子-HS。α-因子-HS肽在酵母细胞停滞测定中具有与真实α-因子相似的生物学效力。α-因子-HS（内酯）还与许多其他化合物反应，包括荧光素、丹酰和生物素的类似物，以产生仅在C-末端衍生的α-因子肽。为了测试表达系统产生较长肽的能力，还产生了编码前因子IX的Gla结构域的60-67个氨基酸残基的肽（FIXQS，FIXQS-His（6））。获得50-55 mg/L纯FIXQS-His（6）和FIXQS-HS（内酯）的产量。
We describe a method to construct tandem repeats of coding sequences for polypeptides interspersed by single methionine residues and terminating in a His(6) tag that can be purified by Ni chelate chromatography and then cleaved by cyanogen bromide (CNBr) into homogeneous peptide units, Annealing and unidirectional ligation of complementary 42mer oligonucleotides encoding the 13 amino acid residue yeast alpha-mating factor (alpha F) followed by ligation of the oligomerized 42mers into a pET vector placed the alpha-factor tandem repeats downstream of the ketosteroid isomerase (KSI) gene and upstream of a His(6) cassette. A KSI-(alpha F)(5)-His(6) fusion was overproduced in Escherichia coli, purified by Ni chelate chromatography, and then cleaved with CNBr to release insoluble KSI, the His(6) tail, and the alpha-factor peptide units, each terminating with homoserine (HS) lactone. HPLC yielded pure peptide in a yield of 56 mg/L. The alpha-factor-HS(lactone) could be ammonolyzed or hydrolyzed to yield alpha-factor-HS-amide or alpha-factor-HS, respectively. The alpha-factor-HS peptide had similar biological potency as authentic alpha-factor in yeast cell arrest assays. The alpha-factor-HS(lactone) was also reacted with a number of other compounds including analogs of fluorescein, dansyl, and biotin to produce alpha-factor peptides derivatized exclusively at the C-terminus. To test the ability of the expression system to produce longer peptides, 60-67 amino acid residue peptides encoding the Gla domain of profactor IX (FIXQS, FIXQS-His(6)) were also produced. Yields of 50-55 mg/L of pure FIXQS-His(6) and FIXQS-HS(lactone) were obtained.