Protein Adsorption on the Nickel-coated Glass Slide for Protein Chips

Hyun, June-Won,Kim, Shi-Yong,Lee, Sang-Hee,Park, Heon-Yong,Pyee, Jae-Ho,Kim, Sung-Hoon Korean Chemical Society 2002 Bulletin of the Korean Chemical Society Vol.23 No.12

The adsorption of proteins on the surface of glass slides is essential for the construction of protein chips. Here, we report that a Histidine (His)-tagged protein protein has been efficiently adsorbed on glass coated with nickel. A variety of nickel chloride-coated plates were prepared by the spin-coating method and adsorbed to the His-tagged protein. When the protein was adsorbed onto the surface of a variety of nickel chloride-coated glass slides, the efficiency of protein adsorption was dependent upon the coating conditions such as nickel chloride concentration, the spin speed and the drying temperature. The slides appropriate for protein adsorption were obtained when the slides were coated with 11%(w/w) of $NiCl_2$ at the spin speed of 4000 rpm for 20 sec and then dried at higher than 40°C. The physical properties of their nickel chloride thin layer were characterized by scanning electron microscopy. x-ray diffraction and atomic force microscopy, finding that the nickel chloride particles were around 10 nm in diameter and uniformly crystallized at 101 faces. These results show that nickel chloride-coated slides prepared by the spin-coating method are utilizable for the construction of Histagged protein chips.

Unfolded Histidine-Tagged Protein is Immobilized to Nitrilotriacetic Acid-Nickel Beads, But Not the Nickel-Coated Glass Slide

Cho Min-Ho,Ahn Sun-Young,Park Heon-Yong Korea Genome Organization 2006 Genomics & informatics Vol.4 No.3

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

Oxygen barrier performance of whey-protein-coated plastic films as affected by temperature, relative humidity, base film and protein type

Hong, Seok-In,Krochta, John M. Elsevier 2006 Journal of food engineering Vol.77 No.3

<P><B>Abstract</B></P> <P>Oxygen permeation properties of whey-protein-coated plastic films were examined to compare their oxygen-barrier performance as affected by temperature (15–40°C), relative humidity (30–85% RH), base film (PE: polyethylene & PP: polypropylene), and protein type (WPI: whey protein isolate & WPC: whey protein concentrate). The resulting whey-protein-coated films showed increase in oxygen permeability (OP) as temperature increased, with an Arrhenius behavior, and activation energy of 10.9–12.1kcal/mol, regardless of types of whey proteins and base films. Relative humidity had an exponential effect on OP of the coated films, with excellent oxygen-barrier properties at low to intermediate RH. Overall OP values of the whey-protein-coated PP were significantly lower than those of the coated PE, mainly due to the intrinsic permeation characteristics of the base films. No remarkable differences could be observed in the oxygen-barrier performance between WPI and WPC coatings. Results suggest that whey protein coatings could work successfully as an oxygen barrier under low moisture conditions and have potential as an alternative to existing expensive synthetic oxygen-barrier polymers in composite structures for food packaging applications.</P>

콜로이드 골드 나노입자의 단백질 수송성 평가법

김미영,노상명,김정목,최한곤,김정애,오유경 한국약제학회 2004 Journal of Pharmaceutical Investigation Vol.34 No.6

Colloidal gold nanoparticles might be of use as nano scale delivery systems of various therapeutic materials in the firture. Recent studies have reported the feasibility of colloidal gold nanoparticles as gene delivery systems or protein delivery systems. In this study, we aimed to develop a short-step method useful for screening the optimal coating conditions of colloidal gold nanoparticles with proteins. We observed that colloidal gold nanoparticles have properties of changing its unique color when they were exposed to NaCl solution. Taking advantage of the color changing properties of colloidal gold nano-particles, we applied the color testing method of colloidal gold nanoparticles solutions for evaluating the protein coating nature. Using bovine senim albumin as a model protein, we tested the protein coating of colloidal gold nanoparticles via the color change upon NaCl addition. The optimal coating concentration and coating conditions of colloidal gold nanoparticles with bovine serum albumin were fixed using the color testing methods. We suggest that the color testing method might be applied to optimize the coating condition of colloidal gold nanoparticles with other therapeutic proteins.

Recombinant Mussel Coating Protein Fused with Cell Adhesion Recognition Motif Enhanced Cell Proliferation

유희영,송영훈,서정현,차형준,황동수 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.2

Endurable coating on biomedical substrates is one of the most important issues in biomedical engineering field. Recently, a robust yet reversible coating has found in the cuticle of mussel byssus and the mimicking of the mussel cuticle coating has been considered as a strategy to build up endurable coating on biomedical materials. To date, the only known protein in the cuticle is mussel foot protein-1 (fp-1). To form endurable and bioactive coating on the biomedical surface, the fusion protein of fp-1 and GRGDSP peptide (fp-1-RGD) was genetically designed and produced in E. coli. The fusion protein of fp-1-RGD was successfully expressed as a form of inclusion body and was simply purified by diluted acetic acid extraction with high purity (~95%). Fp-1-RGD was coated on the tissue culture polystyrene (TCPS) and showed better preosteoblast cell proliferation than that of TCPS. Therefore, the marriage of fp-1 and bioactive peptide can be a good strategy to form bioactive and endurable coating in biomedical field.

TMV 저항성 형질전환 연초식물체 제 5 세대에서 유전자 안정성 및 고온조건에서의 유전자 발현

이기원,박성원,이청호,박은경,김상석,최순용 한국식물생명공학회 1998 식물생명공학회지 Vol.25 No.4

Tobacco mosaic virus(TMV) 외피단백질 유전자를 연초(Nicotiana tabacm cv. NC82)에 형질전환하고 형질전환 식물체 후세대에서 TMV 저항성인 연초를 선발하여, 선발된 TMV 저항성 제5세대 형질전환 식물체의 도입된 유전자발현 및 고온에서의 특성 등을 조사하였다. TMV 저항성 식물체의 염색체 DNA에 TMV 외피 단백질 유전자가 안정되게 존재하고 있음을 genomic PCR을 수행하여 확인하였다. 또한 형질전환 식물체내에서 TMV 외피 단백질 발현은 Immunoblot hybridization 방법으로 확인하였다. TMV 저항성 형질전환 연초식물체에서 발현된 단백질의 양은 매우 적었으며 특히 본엽에는 병징이 나타나지 않았으나 수확기 마지막 액아에 TMV의 반점이 나타난 병징발현 지연형의 형질전환 식물체의 경우에도 발현된 단백질의 양은 정상 NC 82에 TMV가 감염되었을 때와 비교하여 현저히 적었다. TMV 저항성 형질전환 식물체 내에서 발현되는 TMV 외피단백질의 양은 총 단백질에 대비하여 0.01% 이하이였다. TMV 병징 발현 지연형인 형질전환 식물체에 TMV를 인공접종한 후 고온처리상태에서 외피 단백질 유전자의 전사 및 발현을 RT-PCR과 Immune blot hybridization 통하여 확인하였으며, 이때 TMV의 증식도 억제되었으므로 개량멀칭시 나타나는 고온조건하에서도 저항성이 안정적으로 발현될 수 있음을 알 수 있었다. Tobacco mosaic virus(TMV) coat protein cDNA was transformed to Nicotiana tabacum cv. NC82 and the transgenic tobacco plants resistant to TMV infection were isolated in the next generation. The expression of TMV coat protein cDNA and genetic stability of the fifth generation of TMV resistant transgenic tobacco plants at the higher temperature were investigated. The TMV coat protein cDNA was amplified by genomic PCR in all the TMV resistant transgenic tobacco plants. The TMV coat protein expressed in the transgenic tobacco plants was detected at very low level by immunoblot hybridization. Even in tansgenic plants that showed the viral symptom only on very late sucker growth (delay type plants), the coat protein expression in the suckers was much less than that of susceptible tobacco infected with TMV. The TMV coat protein expressed in the transgenic tobacco plants was below 0.01% of total protein. Transcription and expression of the coat protein cDNA in delay type plants were observbed at high temperature (38$^{\circ}C$), and TMV replication was suppressed at both 28$^{\circ}C$ and 38$^{\circ}C$. This indicates that unlike the resistance conferred by 'N' gene. TMV resistance of transgenic tobacco plant won't break down at high temperature.

Surface-Independent Antibacterial Coating Using Silver Nanoparticle-Generating Engineered Mussel Glue

Jo, Yun Kee,Seo, Jeong Hyun,Choi, Bong-Hyuk,Kim, Bum Jin,Shin, Hwa Hui,Hwang, Byeong Hee,Cha, Hyung Joon American Chemical Society 2014 ACS APPLIED MATERIALS & INTERFACES Vol.6 No.22

<P>During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2014/aamick.2014.6.issue-22/am505784k/production/images/medium/am-2014-05784k_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/am505784k'>ACS Electronic Supporting Info</A></P>

Inhibitory effect of soy protein coating formulations on walnut (Juglans regia L.) kernels against lipid oxidation

Kang, H.J.,Kim, S.J.,You, Y.S.,Lacroix, M.,Han, J. Academic Press, etc 2013 LWT- Food science and technology Vol.51 No.1

The aim of this study was to improve the fat stability in walnut (Juglans regia L.) kernels using an edible coating treatment. Coating solutions were composed of soy protein isolate (SPI), carboxymethylcellulose (CMC) and catechin (CT). Walnuts were dipped in coating solution, dried and stored under abuse temperature condition (35 <SUP>o</SUP>C) for 21 days. Lipid oxidation was evaluated by peroxide and thiobarbituric acid reactive substance (TBARS) measurements. Results showed a slight decrease in peroxide values (POV) and a significant reduction of TBARS by coating treatment. The SPI-CT and SPI/CMC-CT coatings were the most effective and decreased the POV by 27 and 31%, respectively, as compared to uncoated walnut after 21 days. The SPI-CT and SPI/CMC-CT coatings also decreased the TBARS value by 16 and 26%, respectively. The incorporation of CT in SPI-based coatings resulted in a synergistic effect on the lipid oxidation preservation. The results of this study show that soy protein-based coating could be a good carrier for antioxidant molecules, and an effective preservative method for extending shelf life and improving the quality stability of oxidation-sensitive kernels.

오이모자이크바이러스 외피단백질유전자 발현 담배의 바이러스 저항성 분석

손성한,김경환,박종석,황덕주,한장호,이광웅,황영수 한국식물생명공학회 1997 식물생명공학회지 Vol.24 No.3

오이 모자이크바이러스(CMV, cucumber mosaic virus)는 작물의 생산량과 품질에 심각한 피해를 주기 때문에 외피단백질 유전자(CP, coat protein gene)를 도입하여 저항성 작물를 개발하고자 하였다. CMV CP유전자가 도입된 형질전환 담배 39 계통을 대상으로 오이모자이크바이러스 저항성을 검정하였다. 바이러스 저항성은 바이러스 감염으로 인한 생장 억제정도, 병징발현에 따른 잎모양의 변화로서 고도저항성, 저항성, 중간성, 감수성 등으로 판정하였고 39개 계통중 16 계통이 뚜렷한 바이러스 저항성을 보였다. 특히, 저항성 계통중 2 계통은 생장량과 잎모양에서 다른 저항성 계통보다 우수하여 고도저항성으로 세분하였다. 각 형질전환계통에서 CP단백질과 CP RNA 생성량을 조사하였는바, CP단백질 생합성은 대부분의 저항성과 감수성계통에서 검출되어 저항성과 특별한 관련을 인정할 수 없었으나 CP RNA는 대부분의 저항성 및 중간성 계통에서 다량 축적되는 경향을 보여 CP RNA가 저항성에 좀더 밀접함을 알수 있었다. 그러나 고도저항성 계통에서는 CP RNA가 검출되지 않아 저항성의 근원을 파악하기 위해서는 계속적인 연구가 요구된다. Cucumber mosaic virus (CMV) leads to a cause of poor crop productivity and quality. To solve this problem, we attempted to develop a virus-resistance tobacco plants by using viral coat protein (CP) gene. Transgenic tobacco plants expressing CMV CP gene were analysed by the resistance upon CMV infection. The virus-resistance was measured in $\textrm{T}_{1}$, generation by the inhibition of plant growth and the expression of the mosaic symptoms infected with CMV. The transgenic lines were divided into four groups: highly resistant, resistant, moderate and susceptible based on their growth and symptom severity. Out of 39 transgenic lines, 16 lines showed significant virus-resistance. And of resistant lines, 2 lines were designated highly resistant based on the facts that they achieved similar plant height to that of non-infected tobacco plants and showed lower disease symptom than that of other lines. The steady state level of CP RNA and coat protein level were measured by northern blot and immunoblot analysis. The CP RNA was highly accumulated in most resistant and moderate lines but barely detected in susceptible lines. The coat protein was detected in most lines regardless of their resistance to CMV. from this result, virus-resistance appeared to correlate more with CP RNA level than the level of coat protein. However, in two highly resistant lines, CP RNA level was unexpectedly low. This unexpected phenomenon need to be further investigated.

Development of Recombinant Coat Protein Antibody Based IC-RT-PCR and Comparison of Its Sensitivity with Other Immunoassays for the Detection of Papaya ringspot virus Isolates from India

M. Sreenivasulu,D. V. R. Sai Gopal 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.2

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus biotype-p (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector,sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E. coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RTPCR)assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. ICRT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.