Membrane protein synthesis: no cells required

Manzer, Zachary A.; Selivanovitch, Ekaterina; Ostwalt, Alexis R.; Daniel, Susan

doi:10.1016/j.tibs.2023.03.006

Cited by 6 publications

(5 citation statements)

References 116 publications

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“… 6 Because enzymes involved in the Kennedy pathway are membrane proteins, it is not easy to reproduce the entire process with recombinant proteins. Cell-free gene expression technologies 7 are able to overcome such difficulty with the use of artificially prepared lipid membrane vesicles (liposomes). 8 Therefore, combining the cell-free system with the fatty acid synthesis system enables synthesizing phospholipids in vitro .…”

Section: Before You Beginmentioning

confidence: 99%

Protocol for in vitro phospholipid synthesis combining fatty acid synthesis and cell-free gene expression

Matsumura,

Sato,

Kuruma

2024

STAR Protocols

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Section: Before You Beginmentioning

confidence: 99%

Protocol for in vitro phospholipid synthesis combining fatty acid synthesis and cell-free gene expression

Matsumura,

Sato,

Kuruma

2024

STAR Protocols

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“…The lack of a cell membrane also allows for an easier manipulation of reaction conditions, including the incorporation of labeled amino acids ( 13 C, 15 N) for massspectrometry or analyzing the effect of cell impermeable drugs. By adding micelles, nanodiscs or organelles into the CFPS system membrane proteins can be studied and puri ed 12,13 . Chinese hamster ovary (CHO) cell lysates together with microsomes have been used to produce CFPS derived antibodies 14 or to produce high yields of di culty to express proteins 15 .…”

Section: Introductionmentioning

confidence: 99%

Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Mansour,

Kipper,

Pulk

2024

Preprint

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We present a highly efficient human HEK293-based cell-free in vitro protein synthesis (CFPS) system that produces 300 µg/ml of reporter protein. The main challenge with the CFPS assay is its limited activity for a few hours, during which all protein is synthesized. If the activity of the CFPS system can be extended, more protein can be produced. The time-dependent inhibition has been studied in the yeast system, but not much is known in the mammalian system. We used the HEK293 CFPS assay to investigate the reasons for time inhibition. We observed that the main culprit is the energy regeneration system, which is depleted quickly. We also demonstrate that the CFPS assay can be used with other mammalian cells or tissues, as evidenced by the active human neuroblastoma SH-SY5Y-based CFPS assay. We observe differences between the yeast and mammalian systems; for example, there is no need to add creatine kinase (CK) as the native CK is functional. This knowledge helps to reduce the costs of CFPS-based systems for biotechnological purposes.

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“…Achievements of the last decades have made cell-free systems (CFs) a good alternative for this aim [14,15]. By adding membrane-like components to CF reaction mixtures, one could achieve co-translational folding of the desired MP [16][17][18], and there are many successful examples of MP production using CFs [19][20][21][22][23][24][25][26]. On the other hand, there is no general approach to synthesizing an arbitrary MP in CFs.…”

Section: Introductionmentioning

confidence: 99%

Facade-Based Bicelles as a New Tool for Production of Active Membrane Proteins in a Cell-Free System

Goncharuk,

Vasileva,

Ananiev

et al. 2023

IJMS

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Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available “Facade” detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system.

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Membrane protein synthesis: no cells required

Cited by 6 publications

References 116 publications

Protocol for in vitro phospholipid synthesis combining fatty acid synthesis and cell-free gene expression

Protocol for in vitro phospholipid synthesis combining fatty acid synthesis and cell-free gene expression

Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Facade-Based Bicelles as a New Tool for Production of Active Membrane Proteins in a Cell-Free System

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