Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli
Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli
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DOI：
10.1002/bit.21396
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发表时间：
2007-10-15
影响因子：
3.8
通讯作者：
Haynes, Charles A.
中科院分区：
文献类型：
--
作者：
Kavoosi, Mojgan;Creagh, A. Louise;Haynes, Charles A.
当家族9碳水化合物结合模块（CBM 9）被插入时，评估接头设计对融合蛋白生产和性能的影响。用作在大肠杆菌中表达的重组蛋白的亲和标签。应用了两种用于接头设计的生物信息学策略：第一种识别宿主生物体的蛋白质组内天然存在的接头，第二种涉及使用生物信息学软件MEROPSTM筛选肽酶及其已知的特异性以设计对宿主内的蛋白质水解具有抗性的人工接头。将使用这些策略设计的接头与传统的聚甘氨酸接头进行比较。虽然广泛使用，但通过串联MS数据发现富含甘氨酸的接头对E.大肠杆菌肽酶。天然（PT）（x）P和MEROPS（TM）设计的S3 N10接头明显更稳定，表明这两种策略提供了一种有用的接头设计方法：因子X，融合蛋白的加工依赖。在接头化学上，聚（G）和S3 N10接头显示出最快的切割速率。发光共振能量转移的研究，用于测量GFP和Tb（III）之间的平均距离的间隔绑定到一个强的钙结合位点的CBM 9，揭示了，对于一个给定的连接器化学，间隔距离随着连接器长度的增加。这种增加对于聚（G）接头特别大，表明这种接头化学采用水合的延伸构型，这使得其特别容易受到蛋白水解的影响。对PT接头系列的差示扫描量热法研究显示，CBM 9与GFP的融合不改变GFP的To，但确实导致不稳定，如通过CBM 9的T-m和Δ H-cal的降低所看到的。不稳定程度增加：随着（PT）（x）P接头长度的减少，使得单个P接头的Delta T-m=-8.4 ° C。
The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9). serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using, the bioinformatics software MEROPS (TM) to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely, used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS (TM)-designed S3N10 linkers were significantly more stable, indicating both strategies provide a useful approach to linker design: Factor X, processing of the fusion proteins depended. strongly on linker chemistry, with poly(G) and S3N10 linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the To, of GFP but did result in a destabilization, as seen by both a decrease in T-m, and Delta H-cal, of CBM9. The, degree of destabilization increased :with decreasing length of the (PT)(x)P linker such that Delta T-m=-8.4 degrees C for the single P linker.