Oliveira Carla, Felix Wagner, Moreira Renato A, Teixeira José A, Domingues Lucília
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Protein Expr Purif. 2008 Aug;60(2):188-93. doi: 10.1016/j.pep.2008.04.008. Epub 2008 May 1.
Frutalin is an alpha-D-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZalphaA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces alpha-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18-20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS-PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide "T-S-S-N", which connects alpha and beta chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the alpha-factor prosequence. Endoglycosidase treatment and SDS-PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-alpha-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62-64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide "T-S-S-N" does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.
菠萝蜜凝集素是一种在面包果种子中表达的α-D-半乳糖结合凝集素。从植物中分离它耗时且会产生不同凝集素亚型的异质混合物。为了提高并便于获取面包果凝集素，我们将优化编码的菠萝蜜凝集素成熟序列克隆到pPICZalphaA表达载体中。这个为在甲基营养型酵母毕赤酵母中进行蛋白表达而设计的表达载体，包含酿酒酵母α因子前导序列，以将重组蛋白导向分泌途径。在培养上清液中检测到了可溶性重组菠萝蜜凝集素，并且它能被天然菠萝蜜凝集素抗体识别。通过尺寸排阻色谱法纯化，从典型的小规模甲醇诱导培养物中每升培养基大约可获得18 - 20毫克重组凝集素。SDS-PAGE和埃德曼降解分析表明，菠萝蜜凝集素以单链蛋白形式表达，因为连接α链和β链的四氨基酸连接肽“T-S-S-N”未被切割。此外，信号序列的不完全加工导致重组菠萝蜜凝集素带有一个源自α因子前导序列的Glu-Ala N端重复序列。内切糖苷酶处理和SDS-PAGE分析表明，重组菠萝蜜凝集素部分进行了N-糖基化。对重组凝集素的进一步表征显示，它特异性结合单糖甲基-α-半乳糖，不过与天然菠萝蜜凝集素相比亲和力较低。从尺寸排阻色谱柱上洗脱下来的重组菠萝蜜凝集素分子量约为62 - 64 kDa，表明其具有四聚体结构，然而它不像天然菠萝蜜凝集素那样能凝集兔红细胞。这项工作表明，半乳糖结合的jacalin相关凝集素的四氨基酸连接肽“T-S-S-N”在毕赤酵母中未发生任何蛋白水解切割，并且连接肽切割对于凝集素糖特异性可能并非必不可少。
