Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasmby Jonghyeok Shin, Young-Hun Jung, Da-Hyeong Cho, Myungseo Park, Kyung Eun Lee, Yoosoo Yang, Cherlhyun Jeong, Bong Hyun Sung, Jung-Hoon Sohn, Jin-Byung Park, Dae-Hyuk Kweon

Enzyme and Microbial Technology


Biotechnology / Applied Microbiology and Biotechnology / Biochemistry


Enzyme and Microbial Technology 79 (2015) 55–62

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Display of membrane proteins on the heterologo by cave

Jonghyeo My

Kyung Eu un

Jin-Byun a Department o iversi b Center for Hu 746, So c Center for The ul 136 d Korea Researc e Department o a r t i c l e i n f o

Article history:

Received 9 February 2015

Received in revised form 20 June 2015

Accepted 26 June 2015

Available onlin




Transmembra a b s t r a c t

Caveolae are membrane-budding structures that exist in many vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicles. Recently, it was shown that caveolae-like structures were formed in Escherichia coli through the expression of caveolin-1. This 1. Introdu

Escherich neered to p the first ho and metabo

E. coli, this eukaryotic metabolic p machinery directly or i ∗ Correspon nology and B

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E-mail add http://dx.doi.o 0141-0229/© e 17 July 2015 caveolae ne protein interesting structure seems to be versatile for a variety of biotechnological applications. Targeting of heterologous proteins in the caveolae-like structure should be the first question to be addressed for this purpose. Here we show that membrane proteins co-expressed with caveolin-1 are embedded into the heterologous caveolae (h-caveolae), the cavaolae-like structures formed inside the cell. Two transmembrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, Syntaxin 1a and vesicle-associated membrane protein 2 (VAMP2), were displayed on the h-caveolae surface. The size of the h-caveolae harboring the transmembrane proteins was ∼100 nm in diameter. The proteins were functional and faced outward on the h-caveolae. Multi-spanning transmembrane proteins FtsH and

FeoB could be included in the h-caveolae, too. Furthermore, the recombinant E. coli cells were shown to endocytose substrate supplemented in the medium. These results provide a basis for exploiting the h-caveolae formed inside E. coli cells for future biotechnological applications. © 2015 Elsevier Inc. All rights reserved. ction ia coli was the first microorganism genetically engiroduce foreign proteins. Since then, E. coli has been st choice for the production of recombinant proteins lic engineering [1]. In spite of the plentiful merits of prokaryote has several disadvantages compared with hosts like Saccharomyces cerevisiae, including limited athways, lower secretion capacity, and poor folding [2,3]. The absence of subcellular organelles in E. coli is ndirectly related to these disadvantages. For example, ding author at: Department of Genetic Engineering, College of Biotechioengineering, Sungkyunkwan University, Suwon 440-746, South 2 31 2907870. ress: dhkweon@skku.edu (D.-H. Kweon). the lack of the Golgi apparatus and endoplasmic reticulum does not allow E. coli to emulate the elegance of eukaryotic protein secretion pathways [4–7]. In addition, the lack of intracellular membrane structure in E. coli is the main cause of insufficient membrane capacity for lipophilic bioproducts and transmembrane proteins.

In a recent study, the formation of heterologous caveolae (hcaveolae) was observed in the cytoplasm of E. coli after the heterologous expression of caveolin-1 [8]. Caveolae are endocytic pits located in mammalian cell surfaces where they play roles in endocytosis, oncogenesis, and the uptake of pathogenic bacteria [9,10]. The h-caveolae formed inside E. coli had been shown to have corresponding size and morphology with those of mammalian cells [8]. Based on these observations, we speculated that the intracellular membrane structures in E. coli is likely to provide several opportunities. First, it may be possible to overexpress active transmembrane proteins inside cells. Though transmembrane proteins are generally accumulated as an inclusion body, rg/10.1016/j.enzmictec.2015.06.018 2015 Elsevier Inc. All rights reserved.olin-1 in the Escherichia coli cytoplasm k Shina,b, Young-Hun Junga,b, Da-Hyeong Choa,b, n Leec, Yoosoo Yangc, Cherlhyun Jeongc, Bong Hy g Parke, Dae-Hyuk Kweona,b,∗ f Genetic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan Un man Interface Nano Technology, Sungkyunkwan University, Suwon, Gyeonggi-do 440ragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seo h Institute of Bioscience & Biotechnology, Daejeon 305-806, South Korea f Food Science and Engineering, Ewha Womans University, Seoul 120-750, South Koreaus caveolae carved ungseo Parka,b,

Sungd, Jung-Hoon Sohnd, ty, Suwon 440-746, South Korea uth Korea -791, South Korea 56 J. Shin et al. / Enzyme and Microbial Technology 79 (2015) 55–62

Fig. 1. Forma me fo

Transmembra (C) Transmissi which is in structures m tein embed proteins ins for metabol estimated t enzymes, b ing the use h-caveolae membrane ble that the surface are products. B molecules i molecules metabolic a demonstrat tested whe caveolin-1 w brane struc of biotechn

To test caveolin-1 2 transmem tor attachm membrane caveolae for was isolated and the actition of recombinant caveolae in E. coli through caveolin-1 expression. (A) A sche ne proteins may be anchored to either plasma membrane or h-caveolae. (B) Endocytosis o on electron microscopy observation of h-caveolae formed in E. coli. Arrows indicate h-ca active protein aggregate, the intracellular membrane ay provide additional space for transmembrane proding. Second, functional expression of transmembrane ide cells may enlarge the number of selectable enzymes ic engineering. About one third of all proteins have been o be membrane-bound, which include cytochrome P450 ut are hardly functionally expressed inside cells limitof such enzymes for metabolic engineering. Third, the may be the first step toward simulating a eukaryotic trafficking pathway in prokaryotes. Fourth, it is possiintracellular membrane structure increases membrane a, which improves the production of lipophilic bioecause perturbation of plasma membranes by lipophilic s expected to be toxic to cells, the isolation of lipophilic in discrete membrane structures may enhance the ctivity and viability of the producer cells. Here, before ing these potential biotechnological versatility, we first ther transmembrane proteins expressed together with ould be functionally targeted to the intracellular memture, which is an essential prerequisite to test the idea ological versatility of the membrane structure. whether membrane proteins co-expressed with are localized to the intracellular membrane structure, brane SNARE (soluble N-ethylmaleimide-sensitive facent protein receptor) proteins, which are part of the fusion machinery, were used as model proteins. The hmed by pinching off from the inner membrane (Fig. 1A), . The orientation of proteins anchored to the h-caveolae vity of the SNARE proteins were tested after characterization of t proteins co formation, h-caveolae 2. Materia 2.1. Expres