Immobilization of His-Tagged Proteins on Various Solid Surfaces Using NTA-Modified Chitosan

Oshige, Masahiko; Yumoto, Kazuhiro; Miyata, Hidefumi; Takahashi, Shunsuke; Nakada, Masaki; Ito, Kanako; Tamegai, Miwa; Kawaura, Hiroki; Katsura, Shinji

doi:10.4236/ojpchem.2013.31002

Cited by 10 publications

(17 citation statements)

References 16 publications

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“…A PMMA microfluidic device channel was functionalised and immobilised with His-tagged enzyme according to protocols adapted from Fixe et al and Oshige et al [[34], [36]]. Briefly, a channel in the PMMA microfluidic device was flushed and filled with isopropanol (99%) and incubated at 30 °C for 3 h. The microfluidic device channel was then rinsed thoroughly with Milli-Q water and incubated with 10% (v/v) hexamethylene-diamine (HMDA) in 100 mM borate buffer pH 11.5, for 2 h. The channel was then thoroughly flushed with Milli-Q water for several channel volumes.…”

Section: Methodsmentioning

confidence: 99%

“…TK was chosen due to its wide substrate tolerance and high enantio- and regio-specificity [28], which make it an attractive biocatalyst for the asymmetric synthesis of chiral metabolites [[29], [30], [31], [32], [33]] and highly relevant in the pharmaceutical industry. However, the method is applicable to a large variety of proteins that can be engineered with a His-tag [34]. The TK-catalysed conversion of hydroxypyruvate (HPA) and glycolaldehyde (GA) for production of the chiral ketoalcohol l -erythrulose (ERY) was chosen as a model reaction (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

et al. 2018

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Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

et al. 2018

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“…A preparation method for protein immobilization substrate reported previously [ 27 ] was slightly modified as follows. Gold coverslips were prepared by attaching gold film (Nilaco, Tokyo, Japan) to glass coverslips (Matsunami, Tokyo, Japan) using adhesive tape (Nichiban, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…Immobilizing large amounts of proteins of interest over a minimal surface area remains challenging for many applications, as does maintaining the orientation of immobilized proteins so as to maintain their activity. To solve the problem of maintaining the proper orientation of immobilized proteins, we have developed protein immobilization methods [ 27 , 28 ] based on specific binding between NTA and the His-tagged protein that was developed by Hochuli et al [ 29 ]. However, most methods including our previously developed methods were immobilized by spotting the purified protein solution.…”

Section: Introductionmentioning

confidence: 99%

A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field

Takahashi

Kishi

Hiraga

et al. 2018

Sensors

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Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

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“…The His-Ni-NTA affinity technique enables the proteins immobilise on a solid surface without altering their function, which has been a challenge in the development of protein assay for decades [63,67]. The use of recombinant affinity tags has addressed the issues of protein orientation and surface density upon immobilisation on solid surface [67,73].…”

Section: Histidine-tagged Membrane Protein Binds To Nickel-nitrilotrimentioning

confidence: 99%

Neutron Reflectometry for Studying Proteins/Peptides in Biomimetic Membranes

Yeung¹,

Shen²

2016

Neutron Scattering

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The development of biomimetic surfaces for protein and peptide adsorptions is continuously expanding. Their biological functions can be influenced by the properties of the underlying artificial environment but the detailed mechanism is still not clear. In the past years, neutron reflectometry has been widely applied to characterise the molecular structure of proteins or multi-protein complexes and their interactions with fluid artificial membrane that mimics the cellular environment. The specific interactions, bindings or structural changes between proteins and membranes play a crucial role in cellular responses and have promising potential in diagnostics and other biosensor applications. This chapter presents the progression of surface design for protein adsorption/interactions on membranes in detail, ranging from a simple phospholipid monolayer setup to more complicated artificial lipid bilayer systems. Furthermore, a new development of designed surfaces for studying the integral membrane protein system is also discussed in this chapter. A brief overview of various membrane mimetic surfaces is first outlined, followed by presenting specific examples of protein-membrane interactions studied by neutron reflectometry. The author demonstrates how to use neutron reflectometry as an advanced technique to provide step-by-step structural details for biomolecular applications in a well-controlled manner.

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Immobilization of His-Tagged Proteins on Various Solid Surfaces Using NTA-Modified Chitosan

Cited by 10 publications

References 16 publications

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field

Neutron Reflectometry for Studying Proteins/Peptides in Biomimetic Membranes

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