Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression

Kyung Yeon Han, Hyuk Seong Seo, Jong Am Song, Keum Young Ahn, Jin Seung Park, Jeewon Lee

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20 Citations (Scopus)

Abstract

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD448-585), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.

Original languageEnglish
Pages (from-to)1536-1543
Number of pages8
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1774
Issue number12
DOIs
Publication statusPublished - 2007 Dec

Bibliographical note

Funding Information:
We thank Professor Hang Chul Shin at Soongsil University for kindly providing the gene clones of mpINS and G-CSF. We also appreciate Professors Won Tae Lee and Hyun Soo Cho at Yonsei University for the kind donation of ppGRN, AID, and Nacht clones. This study was supported by the National Research Laboratory Project (grant no. ROA-2007-000-20084-0) from the Korea Science and Engineering Foundation (KOSEF), funded by the Korean government (MOST) and by the Korea Health 21 R and D Project of the Ministry of Health and Welfare of the Republic of Korea (grant no. A050750). This work was also supported by the Second Brain Korea 21 Project. Additional support for this work from the Korea Science and Engineering Foundation (grant no. R01-2005-000-10355-0), the Korea Research Foundation (grant no. KRF-2004-041-D00180), and the Microbial Genomics and Applications Center (Taejon, Republic of Korea) is also appreciated.

Keywords

  • Crr
  • Escherichia coli BL21 proteome
  • PotD
  • Solubility enhancer
  • Stress response
  • Stress-responsive protein

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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