Part:BBa_K4015014

NusA-Lcp1VH2

NusA-Lcp1VH2 is composed of two basic parts, NusA and Lcp1VH2. NusA is bonded to Lcp1VH2 to enhance the solubility of Lcp1VH2 and in order to increase its yield. NusA is used to enhance the solubility of Lcp through fusing to binding sites of Lcp.

linkage of BBa_K4015001 and BBa_K4015004

In the article, a composite sequence is constructed with Lcp1VH2 and NusA. His6 tag was added due to further purification purpose. In order to increase the overall yield of Lcp in the experiment, we found a fusion partner, which called NusA. The NusA was ligated in pET23a through Spel and Xhal. In general, NusA can work as a solubility enhancer when it is connected with Lcp1VH2, and consequently increase the concentration of Lcp1VH2 produced after being cultivated in E.coli C41::pET23a. In the article, a composite sequence is constructed with Lcp1VH2 and NusA. His6 tag was added due to further purification purpose. The configurations below show the structure of the plasmids.

The effect and result

The solubility of a protein played an important role in the production yields. The previous article brought up a solution, which claims that putting a highly soluble protein to a protein with a low solubility can facilitate protein synthesis. It states that fusing the targeted enzyme to a highly soluble protein can improve the yield of recombinant protein, prevent proteolysis, and increase its solubility.  In our project，our target protein is Lcp1VH2 with a low solubility，an article suggests NusA can act as a fusion partner to improve the production of Lcp1VH2. Therefore, we have inserted NusA as the fusion partner to Lcp, creating a new sequence: NusA-Lcp. Other than increase in solubility led by adding of NusA, we have observed an increase in yield led by NusA. This advantage led by NusA was not expected. Comparing to the original sequence, we have found a significant increase on the productivity of protein once NusA is added, which also benefits the later progression of our project.

https://static.igem.org/mediawiki/parts/4/49/T--KEYSTONE--NusA’s_influence_as_a_fusion_partner.png

Fig 1. NusA’s influence as a fusion partner. SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins NusA-Lcp1VH2 (3). 

unfortunately, the enzyme activity rate of Nus-Lcp1VH2 did not increase comparing to Lcp1VH2, and even decreased. The process for Lcp to degrade rubber requires oxygen consumption. It utilizes the process of β oxidation to break down bonds within polyisoprene. During β oxidation,  Lcp adds two oxygen molecules in between the chemical bonds of polyisoprene. The oxygen consumption rate in the sample tube represents the enzyme activity of Lcp1VH2.  Compared to the sample tube (Supernatant containing Lcp1VH2)，the time for dissolved oxygen dropping to approximately 0mg/l increased from 6 hours to about 16.5 hours in another sample tube (Supernatant containing NusA-Lcp1VH2). This suggests the increase in yield by fusion protein does not increase the enzyme activity.  

Fig 2. The oxygen dissolving test executed on Lcp1VH2 and NusA-Lcp1VH2

Citations:

1. Andler, R., Heger, F., Andreeßen, C., & Steinbüchel, A. (2019). Enhancing the synthesis of latex clearing protein by different cultivation strategies. Journal of Biotechnology, 297, 32-40.

2. Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2020). High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli. Journal of Biotechnology, 309, 92-99. 

3. Amarasinghe C, et al. (2015) The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein Pept Lett 22(10): 885-892.

4. Kapust, R. B., & Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein science : a publication of the Protein Society, 8(8), 1668–1674. https://doi.org/10.1110/ps.8.8.1668 

5. Schmidt, M. C., & Chamberlin, M. J. (1987). nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. Journal of molecular biology, 195(4), 809–818.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1371
  Illegal XhoI site found at 2001
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1620
  Illegal NgoMIV site found at 1978
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1605
  Illegal SapI.rc site found at 1593