Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombi...Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.展开更多
Food allergens are mainly naturally-occurring proteins with immunoglobulin E(IgE)-binding epitopes.Understanding the structural and immunogenic characteristics of allergenic proteins is essential in assessing whether ...Food allergens are mainly naturally-occurring proteins with immunoglobulin E(IgE)-binding epitopes.Understanding the structural and immunogenic characteristics of allergenic proteins is essential in assessing whether and how food processing techniques reduce allergenicity.We here discuss the impacts of food processing technologies on the modification of physicochemical,structural,and immunogenic properties of allergenic proteins.Detection techniques for characterizing changes in these properties of food allergens are summarized.Food processing helps to reduce allergenicity by aggregating or denaturing proteins,which masks,modifies,or destroys antigenic epitopes,whereas,it cannot eliminate allergenicity completely,and sometimes even improves allergenicity by exposing new epitopes.Moreover,most food processing techniques have been tested on purified food allergens rather than food products due to potential interference of other food components.We provide guidance for further development of processing operations that can decrease the allergenicity of allergenic food proteins without negatively impacting the nutritional profile.展开更多
CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in c...CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild展开更多
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer m...Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.展开更多
Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms un...Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes.Furthermore,the principle of protein assembly gives guidelines for new biomimetic materials with potential appli-cations in medicine,energy,and nanotechnology.Atomic force microscopy(AFM)is a powerful tool for investigating protein assembly and interactions across spatial scales(single molecules to cells)and temporal scales(milliseconds to days).It has significantly contributed to understanding nanoscale architectures,inter-and intra-molecular interactions,and regulatory elements that determine protein structures,assemblies,and functions.This review describes recent advancements in elucidating protein assemblies with in situ AFM.We discuss the structures,diffusions,interac-tions,and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging,bimodal imaging,live cell imaging,and machine-learning-enhanced data analysis.These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.展开更多
文摘Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.
基金supported by the National Natural Science Foundation of China (32102605)the Agricultural Science and Technology Innovation Program under Grant (CAAS-ASTIP-2020IAR)the Earmarked Fund for CARS (CARS-44)。
文摘Food allergens are mainly naturally-occurring proteins with immunoglobulin E(IgE)-binding epitopes.Understanding the structural and immunogenic characteristics of allergenic proteins is essential in assessing whether and how food processing techniques reduce allergenicity.We here discuss the impacts of food processing technologies on the modification of physicochemical,structural,and immunogenic properties of allergenic proteins.Detection techniques for characterizing changes in these properties of food allergens are summarized.Food processing helps to reduce allergenicity by aggregating or denaturing proteins,which masks,modifies,or destroys antigenic epitopes,whereas,it cannot eliminate allergenicity completely,and sometimes even improves allergenicity by exposing new epitopes.Moreover,most food processing techniques have been tested on purified food allergens rather than food products due to potential interference of other food components.We provide guidance for further development of processing operations that can decrease the allergenicity of allergenic food proteins without negatively impacting the nutritional profile.
文摘CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild
基金Supported by the Key Laboratory Foundation of the Educational Department of Liaoning Province (No. 2009S024)the Dalian Municipal Government of China (No. 2007B11NC069)the Grant of Dalian Ocean University (No. SY2007005)
文摘Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.
基金National Natural Science Foundation of China,Grant/Award Numbers:32371525,T2221001,92353304,T2350011Strategic Priority Research Program of the Chinese Academy of Sciences,Grant/Award Number:XDB37020105+5 种基金U.S.Department of EnergyOffice of ScienceOffice of Basic Energy Sciences,Grant/Award Number:FWP 65357Pacific Northwest National LaboratoryEnergy Frontier Research CentersCenter for the Science of Synthesis Across Scales,Grant/Award Number:DE-SC0019288。
文摘Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes.Furthermore,the principle of protein assembly gives guidelines for new biomimetic materials with potential appli-cations in medicine,energy,and nanotechnology.Atomic force microscopy(AFM)is a powerful tool for investigating protein assembly and interactions across spatial scales(single molecules to cells)and temporal scales(milliseconds to days).It has significantly contributed to understanding nanoscale architectures,inter-and intra-molecular interactions,and regulatory elements that determine protein structures,assemblies,and functions.This review describes recent advancements in elucidating protein assemblies with in situ AFM.We discuss the structures,diffusions,interac-tions,and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging,bimodal imaging,live cell imaging,and machine-learning-enhanced data analysis.These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.
