High expression of Fightless I (FLII) is associated to multiple tumors. Based on our previous study that FLII might be involved in the nuclear export, we assessed the possible interaction of FLII with the nuclear envelop associating proteins Importin β and Nup88. We first constructed GST-FLII, GST-LRR recombinant plasmids and transformed them into the Rosetta strain to produce GST-FLII, GST-LRR fusion protein. After purification of these proteins, GST-pull down, as well as co-immunoprecipitation, were used to test the interaction of FLII with Importin β and Nup88. FLII interacted with Importin β and Nup88, and FLII LRR domain is responsible for these interactions. Thus, FLII may play a role in nuclear export through interaction with Importin β and Nup88.
Humans ; Microfilament Proteins ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Recombinant Fusion Proteins ; metabolism ; beta Karyopherins ; metabolism

The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.
Animals ; Bombyx ; genetics ; metabolism ; Chitin ; metabolism ; Insect Proteins ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; Silk ; biosynthesis

OBJECTIVETo study the influence of C-terminal domain of ADAMTS13 on its cleaving activity.

METHODSThe full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF.

RESULTSThe recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress.

CONCLUSIONThe distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.

Galium ; Humans ; Recombinant Proteins ; metabolism ; Transfection ; von Willebrand Factor ; genetics

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Enzymes/metabolism* ; Recombinant Proteins/genetics*

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2, 6-bisphosphate (Fru-2, 6-P₂), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28 ℃, respectively. The purified sin[A1] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.
Humans ; Carica/genetics* ; Recombinant Proteins ; Carbohydrate Metabolism ; Cloning, Molecular ; China

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K_m and k_cat/K_m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.
Recombinant Proteins/metabolism* ; Paenibacillus/metabolism* ; Mannose/metabolism* ; Escherichia coli/metabolism* ; Mannitol/metabolism*

OBJECTIVETo investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.

METHODSThe PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.

RESULTSThe transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.

CONCLUSIONSoluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Escherichia coli ; metabolism ; Genetic Vectors ; Hepacivirus ; Recombinant Proteins ; genetics ; metabolism ; Viral Core Proteins ; biosynthesis ; genetics ; metabolism ; Viral Nonstructural Proteins ; metabolism

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.
Animal ; Baculoviridae/genetics ; Blotting, Western ; Cell Line ; Factor VIII/metabolism* ; Factor VIII/genetics ; Factor VIII/chemistry ; Factor VIII/biosynthesis ; Genetic Vectors ; Human ; Mutagenesis, Site-Directed ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/chemistry ; Recombinant Proteins/biosynthesis ; Spodoptera

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Paenibacillus ; enzymology ; Recombinant Proteins ; genetics ; gamma-Cyclodextrins ; metabolism

The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a (+) and transformed into host Escherichia coli strain BL21 (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.
Animals ; Cattle ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Ghrelin ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism