Zblewska Kamila, Krajewska Joanna, Zolkiewski Michal, Kędzierska-Mieszkowska Sabina
Department of Biochemistry, University of Gdansk, 80-308 Gdansk, Poland.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
Arch Biochem Biophys. 2014 Aug;555-556:23-7. doi: 10.1016/j.abb.2014.05.012. Epub 2014 Jun 2.
Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria.
在细菌系统中过量生产异源蛋白通常会导致不溶性包涵体（IBs）的形成，这是生化研究和生物技术中的一个主要障碍。原则上，可以利用分子伴侣的活性来控制包涵体的形成并提高重组蛋白产量，但主要细菌伴侣（DnaK/J、GroEL/ES和ClpB）处理包涵体的潜力尚未完全确定。我们研究了在存在和不存在伴侣蛋白ClpB的情况下，在大肠杆菌中过量生产的两种易于聚集的蛋白VP1LAC和VP1GFP包涵体的形成情况。我们发现，在包涵体产生过程中，两种ClpB亚型ClpB95和ClpB80都在大肠杆菌细胞中积累。在没有ClpB的情况下，包涵体蛋白的量增加。ClpB支持大肠杆菌细胞中聚集的VP1LAC和VP1GFP的复性和再激活。在ClpB95和ClpB80同时存在的情况下，包涵体解聚效果最佳。我们的结果表明ClpB在控制细菌中的蛋白质聚集和包涵体形成方面起着至关重要的作用。
