Spencer J A, Jeenes D J, MacKenzie D A, Haynie D T, Archer D B
Institute of Food Research, Norwich Research Park, Colney, UK.
Eur J Biochem. 1998 Nov 15;258(1):107-12. doi: 10.1046/j.1432-1327.1998.2580107.x.
Fusion proteins are used to enhance the yields of heterologous proteins secreted from filamentous fungi. In Aspergillus niger, the target protein is normally fused downstream of the carrier protein glucoamylase with a Lys-Arg KEX2-like cleavage site at the junction. This is cleaved in vivo to release mature protein but the processing is not always accurate. We have used N-terminal mutant lysozymes to vary the sequence immediately downstream of the KEX site, and also varied the amino acid sequence upstream of the KEX processing site, to study the fidelity of processing. The sequences both upstream and downstream of the KEX2 site affected the fidelity of cleavage. With some constructs, a range of processing sites were apparent and the relative proportions were time dependent in batch cultures of A. niger. Aberrant processing was related to the secondary-structure preferences of the amino acids in and around the KEX site. Downstream of the processing site, the fidelity of processing decreased in proportion to the tendency for helix formation.
融合蛋白用于提高丝状真菌分泌的异源蛋白产量。在黑曲霉中，目标蛋白通常与载体蛋白糖化酶融合在其下游，连接处有一个类似KEX2的赖氨酸-精氨酸切割位点。该位点在体内被切割以释放成熟蛋白，但加工过程并不总是准确的。我们使用N端突变溶菌酶来改变KEX位点下游紧邻的序列，同时也改变KEX加工位点上游的氨基酸序列，以研究加工的保真度。KEX2位点上下游的序列均影响切割的保真度。对于某些构建体，一系列加工位点很明显，并且在黑曲霉的分批培养中，相对比例与时间有关。异常加工与KEX位点及其周围氨基酸的二级结构偏好有关。在加工位点下游，加工保真度的降低与螺旋形成趋势成比例。
