A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library

Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes.     

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.     

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (R _gfp/A _xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R _gfp/A _xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.     

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).     

A yeast platform for the production of single-chain antibody-green fluorescent protein fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner

Human interleukin-2 (hIL-2) production in Escherichia coli and insect cell/baculovirus expression systems can be inefficient. Here we investigated secreted production of hIL-2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogaster S2 cells. This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins. We report that, following stable transfection and induction, S2 cells secreted hIL-2 as a fusion protein (approximately 2.3 microg/mL yield), with a secretion efficiency of approximately 90%. Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL-2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system. The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL-2 expression.     

Effect of an amyloidogenic sequence attached to yellow fluorescent protein

Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence. Seven fusion proteins formed white fibrils lacking native YFP function. However, the fusion with an 18-residue medin sequence and a 50 amino acid linker formed fibrils with yellow color of folded YFP. Deconvolution analysis of infrared spectra also supports the presence of properly folded YFP in the fibrils formed by this protein. These results suggest that, the presence of an amyloidogenic sequence to a folded protein can promote the formation of fibrils and disrupt the native structures whereas the structure of the folded region is retained by optimizing sequences of amyloidogenic and linker regions.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Diversity of the CYP21P-like gene in CYP21 deficiency

More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene. The occurrence of defective CYP21 genes, including 15 mutations, has been attributed to intergenic recombination of DNA sequences from CYP21P, and shows no influence on the RP1-C4A-CYP21P-XA-RP2-C4BCYP21- TNXB gene locus on chromosome 6p21.3. However, multiple gene deletions in this region produce at least three categories of gene arrangements: (a) C4A-CYP21P/CYP21-TNXB, in which there is a CYP21P/CYP21 fusion gene; (b) C4A-XCYP21-TNXB, where XCYP21 indicates that the CYP21 gene contains mutations of IVS2 (-12A/C>G and 707-714delGAGACTAC); and (c) C4A-CYP21P-TNXA/TNXB, in which the TNX A and B genes are fused. Among them, seven different structures of the CYP21 haplotype were found at these three loci. Formation of the C4A-CYP21P/CYP21-TNXB locus produced four distinct CYP21P/CYP21 chimeras. The C4A-XCYP21-TNXB locus contained the IVS2 mutation -12A/C>G and 707-714delGAGACTAC from the XCYP21 gene; and two kinds of TNXA/TNXB hybrids were found in the C4A-CYP21P-TNXA/TNXB locus. The seven different CYP21 alleles produced 3.2 kb Taq I fragments caused by deletion of the RP2-XA-C4B locus. Therefore, production of a 3.2-kb CYP21 allele shows diversity, but is not a unique feature of the CYP21P gene. Most of these gene arrangements probably exist in the C4A-XCYP21-TNXB and C4A-CYP21P/CYP21-TNXB gene loci. The existence of the C4A-CYP21P-TNXA/TNXB locus might not be common in CAH patients with 21-hydroxylase deficiency.     

过表达TRAF2降解外源性绿色荧光蛋白

TRAF2是机体免疫与炎症反应中具有重要作用的蛋白,有E3连接酶活性;GFP是一种受到激发光照射后可产生绿色荧光的蛋白,常用于蛋白质的细胞共定位研究,并显示与GFP融合表达靶蛋白在细胞中的位置．我们实验发现共转染表达质粒pCMV-myc-TRAF2与pEGFP-C3,可引起转染细胞绿色荧光减弱．Western印迹实验证明TRAF2可以降解GFP,并且这种降解是通过蛋白酶体途径进行的．为进一步确定这种降解的特异性,在HEK293细胞中共转pCMV-myc-TRAF2与pEGFP-C3-LNX后,发现随着TRAF2表达量增加,融合蛋白GFP-LNX减少;而共转质粒pCMV-myc-TRAF2与pCMV-myc-LNX后,未发现LNX蛋白表达减少,表明TRAF2对GFP的降解具有相对特异性．GFP是人类细胞中并不存在的蛋白,TRAF2能够将其降解可能意味着TRAF2参与了细胞抗病原体感染的过程．     

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.     

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.     

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer

Tissue-like structures of cells organized in vitrohave a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices. This revised version was published online in August 2006 with corrections to the Cover Date.     

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.     

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)₃ linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

Targeting expression of the catalytic domain of the kinase insert domain receptor (KDR) in the peroxisomes of Pichia pastoris

The kinase insert domain receptor (KDR), also known as vascular endothelial growth factor receptor-2 (VEGFR2), is an important therapeutic target for the treatment of cancer because of its crucial role in angiogenesis, which is fundamental to the malignancy of tumors. Here, we expressed the catalytic domain of KDR in Pichia pastoris under the control of the AOX1 promoter. In order to facilitate its purification and detection, His-tag and green fluorescent protein (GFP) were fused to the N-terminus of KDR. At the same time, a peroxisomal targeting signal 1 (SKL) was fused to the C-terminus to avoid the potential negative effect on the host cell. The highly expressing clone K1 was selected by GFP fluorescence intensity analysis using flow cytometry (FCM). Furthermore, the GFP-KDR-SKL fusion protein was proved to be correctly targeted to the peroxisomes of P. pastoris by colocation with blue fluorescent protein-SKL. The expression of GFP-KDR-SKL led to extensive phosphorylation of endogenous proteins and significantly inhibited cell growth. However, the expression was not lethal to the cells. Both in vitro biological activity assay and inhibition rate assay demonstrated that the purified GFP-KDR-SKL fusion protein exhibited high kinase catalytic activity and could be used as a target for anticancer drug screening.     

The role of quarternary structure in fluorescent protein stability

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.     

绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡