Collén Anna, Selber Klaus, Hyytiä Teppo, Persson Josefine, Nakari-Setlä Tiina, Bailey Michael, Fagerström Richard, Kula Maria-Regina, Penttilä Merja, Stålbrand Henrik, Tjerneld Folke
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Sweden.
Biotechnol Bioeng. 2002 May 20;78(4):385-94. doi: 10.1002/bit.10232.
Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
在此，我们展示数据以证明通过向蛋白质分子添加短链氨基酸残基，亲水性酶如何能够被导向水相双相系统中富含疏水去污剂的相。目标酶是里氏木霉工业上重要的内切葡聚糖酶I，EGI（内切-1,4-β-D-葡聚糖-4-葡聚糖水解酶，EC 3.2.1.4，Cel7B）。我们研究了三种含有不同C末端肽延伸（包括不同长度和定位的色氨酸-脯氨酸基序）的EGI变体的分配情况。此外，利用最近开发的由热分离共聚物HM-EOPO组成的系统来研究融合标签的作用。添加含色氨酸残基的肽增强了EGI向富含HM-EOPO相的分配。由非离子去污剂（Agrimul NRE1205）组成的系统，对于含有短连接子延伸的构建体EGI(core-P5)(WP)(4)（含有(色氨酸-脯氨酸)(4)），产生了最高的分配系数（K = 31）和产率（90%）。构建了用于大规模生产的里氏木霉Rut-C30重组菌株，其中融合蛋白EGI(core-P5)(WP)(4)由纤维素酶基因cbh1的强启动子表达。融合蛋白在摇瓶培养过程中成功地从真菌中表达并分泌出来。然而，在28-L生物反应器中的培养表明，融合蛋白对蛋白酶敏感。因此，在大规模生产试验中仅获得了低产量水平。
