Mohanty Shilpa, Dabburu Govinda Rao, Kumar Manish, Khasa Yogender Pal
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Biophysics, University of Delhi South Campus, New Delhi 110021, India.
Int J Biol Macromol. 2022 Jun 1;209(Pt A):1001-1019. doi: 10.1016/j.ijbiomac.2022.04.078. Epub 2022 Apr 18.
Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% α-helices, 36.7% β-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD T was found to be 45 °C. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with Y of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h.
小泛素相关修饰物（SUMO）蛋白被有效地用于在大肠杆菌中靶向各种难以表达的蛋白的可溶性表达。然而，其在大规模蛋白质生产中的应用受到Ulp成本较高的限制，Ulp是从目标蛋白上切割SUMO融合标签以产生真实N端所必需的。本研究鉴定并表征了两种新型SUMO蛋白酶，即来自粟酒裂殖酵母的Ulp1和Ulp2。对密码子优化的基因序列进行克隆并在大肠杆菌中表达。使用生物信息学工具推导了SpUlp1和SpUlp2催化结构域的序列和结构。蛋白质-蛋白质相互作用研究预测，与酿酒酵母（ScUlp1）的对应物相比，SpUlp1对SUMO具有更高的亲和力。使用Ni-NTA色谱法纯化SpUlp1的催化结构域，回收率为83.33%。此外，体外活性数据进一步证实了SpUlp1催化结构域的快速作用特性，孵育1小时内融合蛋白的切割率达到90%，表明粟酒裂殖酵母Ulp1的新颖性和商业相关性。生物物理表征显示SpUlp1SD中α螺旋占8.8%，β折叠占36.7%。从热圆二色性和荧光数据来看，SpUlp1SD的Tm为45℃。此外，使用补料分批培养进行生物工艺优化，SpUlp1SD的产量为3.5 g/L，产率为77.26 mg/g DCW，体积生产力为205.88 mg/L/h。
