Atomic force microscopy is a rather new type of nano microscopic technology. It has some advantages, such as high resolution (sub-nano scale); avoidance of special sample preparation; real-time detection of samples under nearly physiological environment; in situ study of samples under water environment; feasibility of investigating physical and chemical properties of samples at molecular level, etc. In recent years, the application of atomic force microscopy in protein study has brought about outstanding achievements. In this paper are introduced the principle and operation modes of atomic force microscopy, also presented are its application in protein imaging, adsorption, folding-and-unfolding, assembly, and single molecular recognition. Additionally, the future application of atomic force microscopy in protein study is prospected.
Animals ; Humans ; Microscopy, Atomic Force ; Protein Conformation ; Protein Folding ; Protein Unfolding ; Proteins ; chemistry ; ultrastructure

A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E. coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation, After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demonstrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.
Chromatography, Gel ; Circular Dichroism ; Metalloendopeptidases ; chemistry ; isolation & purification ; Oligopeptides ; isolation & purification ; Protein Folding ; Protein Renaturation ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

OBJECTIVETo characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation.

METHODSThe refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.

RESULTSRenaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.

CONCLUSIONThe degree of protein renaturation correlated with the IgE-binding capacity of the protein. Results from this study may be of help for food allergy therapy and development of vaccination in the future.

Allergens ; Animals ; Cattle ; Circular Dichroism ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin E ; Lactoglobulins ; chemistry ; metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Denaturation ; Protein Folding ; Spectrometry, Fluorescence ; methods

The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.
Antibodies, Catalytic ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Bioreactors ; microbiology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Immunoglobulin Fragments ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Inclusion Bodies ; metabolism ; Protein Folding ; Protein Renaturation ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Selenium ; metabolism

Artificial intelligence (AI), big data, and ubiquitous robotic companions —the three most notable technologies of the 4th Industrial Revolution—are receiving renewed attention each day. Technologies that can be experienced in daily life, such as autonomous navigation, real-time translators, and voice recognition services, are already being commercialized in the field of information technology. In the biosciences field in Korea, such technologies have become known to the local public with the introduction of the AI doctor Watson in large number of hospitals. Additionally, AlphaFold, a technology resembling the AI AlphaGo for the game Go, has surpassed the limit on protein folding predictions—the most challenging problems in the field of protein biology. This report discusses the significance of AI technology and big data on the bioscience field. The introduction of automated robots in this field is not just only for the purpose of convenience but a prerequisite for the real sense of AI and the consequent accumulation of basic scientific knowledge.
Artificial Intelligence ; Biology ; Biotechnology ; Friends ; Humans ; Korea ; Protein Folding ; Voice

OBJECTIVETo study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF).

METHODSSOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay.

RESULTSThe expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively.

CONCLUSIONThis research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.

Animals ; Arginine ; chemistry ; genetics ; metabolism ; Base Sequence ; Biological Assay ; Cell Proliferation ; Chromatography, Affinity ; Chromatography, Ion Exchange ; Cytokines ; metabolism ; Escherichia coli ; genetics ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; genetics ; isolation & purification ; metabolism ; Humans ; Interleukin-2 ; chemistry ; genetics ; isolation & purification ; metabolism ; Mice ; Molecular Sequence Data ; Protein Folding ; Protein Renaturation ; Recombinant Fusion Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

Neurodegenerative diseases are a group of chronic progressive neuronal damage disorders. The cause is unclear, most of them share a same pathological hallmark with misfold proteins accumulating in neurons. Carboxyl-terminus of Hsc70 interacting protein (CHIP) is a dual functional molecule, which has a N terminal tetratrico peptide repeat (TPR) domain that interacts with Hsc/Hsp70 complex and Hsp90 enabling CHIP to modulate the aberrant protein folding; and a C terminal U-box ubiquitin ligase domain that binds to the 26S subunit of the proteasome involved in protein degradation via ubiqutin-proteasome system. CHIP protein mediates interactions between the chaperone system and the ubiquitin-proteasome system, and plays an important role in maintaining the protein homeostasis in cells. This article reviews the molecular characteristics and physiological functions of CHIP, and its role in cellular metabolism and discusses the relationship between CHIP dysfunction and neurodegenerative diseases.
Animals ; Humans ; Neurodegenerative Diseases ; genetics ; metabolism ; Protein Binding ; Protein Folding ; Proteolysis ; Ubiquitin-Protein Ligases ; genetics ; metabolism

Catalysis ; Diffusion ; Magnetic Resonance Spectroscopy ; methods ; Molecular Dynamics Simulation ; Protein Conformation ; Protein Folding ; Proteins ; chemistry

The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.
Lipase/metabolism* ; Molecular Chaperones/metabolism* ; Protein Folding ; Protein Precursors ; Substrate Specificity

The mechanism of oxidative refolding of proteins was elucidated in more detail from the intensive and extensive studies in the past decades. 1. Most of the proteins examined so far proceed oxidative refolding via multiple pathways rather than a single and specific pathway. This is consistent with the folding energy landscape theory. 2. It is the native interactions rather than the non-native interactions that direct the folding process. This is not necessarily incompatible with the importance of the non-native disulfide intermediates in the bovine pancreatic trypsin inhibitor (BPTI) pathway, which are just a chemical necessity in the intramolecular arrangement to facilitate native disulfide formation. 3. Based on the BPTI refolding it was suggested that disulfide bonds have a stabilizing effect on the native state without determining either the folding pathway or the final three-dimensional structure of the protein. This point of view is not applicable to other proteins. Studies on the refolding of prochymosin unequivocally demonstrated that the formation of native disulfides is the prerequisite to the recovery of the native conformation. It is more likely that the interdependence between the native disulfide formation and the formation of native structure is a general rule. 4. At the early stage of oxidative refolding disulfide formation is essentially a random process, with the progress of refolding further disulfide formation is increasingly dependent on the conformations of the intermediates. Enhancing the renaturation yield of recombinant proteins is a major challenge in biotechnology. In addition to aggregation, the formation of species with mispaired disulfide bonds is a leading cause of decreased yield. Progress in understanding the mechanism of oxidative refolding has provided insight into how to solve this problem. As described above, at the later stage of refolding disulfide formation depends on the conformations of intermediates. The intermediates with native-like and flexible structure favourable for native disulfide formation and correct refolding are productive intermediates, while the unproductive intermediates tend to adopt stable conformations, which render the thiol groups and disulfide bond(s) inaccessible and further folding unfavourable energetically. Therefore, the principle to enhance the renaturation yield of disulfide-containing proteins is to cause the productive intermediates to predominate by destabilizing the unproductive intermediates. To approach this, alkaline pH, low temperature, labilizing agents, protein disulfide isomerase and its analogues and alteration of primary structure have been proved useful to adjusting the structure of the unproductive intermediates so as to facilitate thiol/disulfide interchange and in turn the native disulfide formation. The prospects for the oxidative refolding of proteins both in basic and applied researches are discussed in this review article.
Animals ; Biotechnology ; Disulfides ; chemistry ; Humans ; Oxidation-Reduction ; Protein Folding ; Proteins ; chemistry ; genetics ; metabolism