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.     

Thermodynamic and computational studies of DNA triple helices containing a nucleotide or a non-nucleotide linker in the third strand.

In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)(m)(pyrimidine)(m)-3'. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3'-3' phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.     

重组单链抗体-低分子质量尿激酶融合蛋白在 CHO 细胞中的抗降解研究

抗人纤维蛋白单链抗体-低分子质量尿激酶(Ⅱn-UK)融合蛋白,兼有单链抗体对纤维蛋白的亲和性和尿激酶的溶栓活性,有望开发成为新型导向溶栓药物.但基于通用连接肽(G4S)3的Ⅱn-linker-UK融合蛋白在CHO细胞中表达时出现明显的降解.为了解决此问题,利用分子生物学方法,对Hn-UK融合蛋白进行了分子改造,包括置换连接肽,改变两个半分子(moiety)的相对位置,以及对连接肽附近明确的蛋白酶位点进行突变等方法,并分别研究了改造后的11种Ⅱn-1inker-UK或UK-linker-Ⅱn突变体在CHO细胞中分泌性表达时的稳定性,最终筛选到一种抗降解的突变体.     

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.     

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis,Biophysical Measurements,and Molecular Simulation

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.     

An analysis of protein domain linkers: their classification and role in protein folding

Recent advances in protein engineering have come from creating multi-functional chimeric proteins containing modules from various proteins. These modules are typically joined via an oligopeptide linker, the correct design of which is crucial for the desired function of the chimeric protein. Here we analyse the properties of naturally occurring inter-domain linkers with the aim to design linkers for domain fusion. Two main types of linker were identified; helical and non-helical. Helical linkers are thought to act as rigid spacers separating two domains. Non-helical linkers are rich in prolines, which also leads to structural rigidity and isolation of the linker from the attached domains. This means that both linker types are likely to act as a scaffold to prevent unfavourable interactions between folding domains. Based on these results we have constructed a linker database intended for the rational design of linkers for domain fusion, which can be accessed via the Internet at http://mathbio.nimr.mrc.ac.uk.     

Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers

The flexible peptides (GGGGS)n (n < or = 3), the alpha-helical peptides (EAAAK)n (n < or = 3) and two other peptides were used as linkers to construct bifunctional fusions of beta-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both beta-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304-426% and 82-143% of the parental ones, respectively. The peptide linker (GGGGS)(2) resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)(3) also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.     

Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization

Many plant gums, such as gum arabic, contain hydroxyproline-rich glycoproteins (HRGPs), which are also abundant components of the plant cell extracellular matrix. Here we expressed in transgenic BY2 Nicotiana tabacum (tobacco) cells, a synthetic gene encoding a novel HRGP-based gum, designated gum arabic-8 or (GA)(8). (GA)(8) encoded eight repeats of the consensus polypeptide sequence of gum arabic glycoprotein (GAGP): Gly-Pro-His-Ser-Pro-Pro-Pro-Pro-Leu-Ser-Pro-Ser-Pro-Thr-Pro-Thr-Pro-Pro-Leu, in which most of the Pro residues were posttranslationally modified to hydroxyproline (Hyp). (GA)(8) was expressed as a green fluorescent protein (GFP) fusion protein targeted to the culture medium, (GA)(8)GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)(8)GFP was cell growth-associated. The extracellular yield of (GA)(8)GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)(8)GFP was purified from the culture medium by a combination of hydrophobic interaction, gel permeation, and reversed phase chromatography. Biochemical characterization indicated that the amino acid composition of the (GA)(8) module, after removal of GFP by proteolysis, was virtually identical to that of predicted by the GAGP consensus sequence and that carbohydrate, which occurred as arabinogalactan polysaccharides and small oligoarabinosides O-linked through the Hyp residues, accounted for 84% of the molecules' dry weight. Functional assays showed that (GA)(8) exhibited low viscosity in aqueous solution similar to native GAGP. However, neither GFP alone nor the (GA)(8) module could emulsify orange oil. However, the fusion protein (GA)(8)GFP possessed 1.28-fold better emulsification properties than native GAGP. This work demonstrates the feasibility and potential of a synthetic gene approach to the de novo design of novel glycoprotein-based gums and emulsifiers.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.     

Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha2b fusion protein by linker engineering

Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha2b (IFN-alpha2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha2b was ascribed to the structural disturbance between HSA and IFN-alpha2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.     

Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes

A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g x l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degrees C during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g x l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.     

The MURFI linker for multiple reading frame insertion of a sense or nonsense codon into DNA.

Blunt-end palindromic DNA linkers with a central restriction site have been designed for the multiple reading frame insertion (abbreviated MURFI) of a sense or nonsense codon into DNA. We have utilized an amber MURFI linker, 5'CTAG TCTAGA CTAG3' to disrupt the lacZ gene, yielding truncated beta-galactosidase proteins. Conditional disruption of the tetr gene in E. coli has also been demonstrated. Nonsense codon MURFI linkers permit conditional fusion of multiple gene products while sense codon linkers can add structural elements (e.g. beta-turn, cationic segment, hydrophobic segment) or a desired amino acid to a protein (e.g. methionine, cysteine). Shotgun or alternatively site-directed insertion of the symmetric linkers is possible. The over-all length of the linker may be adjusted to retain the original reading frame, matching nucleotide additions or subtractions at recipient DNA sites. If a linker restriction site occurs elsewhere in the target DNA, single linker copies may still be inserted using non-phosphorylated linkers.     

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m,a novel potent CD4-antibody fusion protein against HIV-1

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.     

The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.     

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.     

Search for the optimal linker in tandem hairpin polyamides

In order to target specific DNA sequences >or=10 base pairs in size by minor groove binding ligands, a search for the optimal linker in dimers of hairpin polyamides was initiated. Two series of tandem polyamides ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-L](HN)gamma-PyPyPy-beta-Dp (1a-e), where L represents a series of 4-8 carbon long aliphatic amino acid linkers, and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-L](HN)gamma-PyPyPy-beta-Dp (2a-e), where L represents a series of 2-6 carbon long aliphatic amino acid linkers, were synthesized and characterized by quantitative DNase I footprinting. beta, gamma and Dp represents beta-alanine, gamma-aminobutyric acid, and 3-(dimethylamino)propylamine, respectively. It was found that the five-carbon 5-aminovaleric acid (delta), is suitable to span one base-pair (bp) of DNA when incorporated into a tandem polyamide. ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-delta](HN)gamma-PyPyPy-beta-Dp (1b) binds the 10 bp binding-site 5'-AGTGAAGTGA-3' with equilibrium association constant K(a)=3.2 x 10(10) M(-1) and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-delta](HN)gamma-PyPyPy-beta-Dp (2d) binds the 11 bp binding-site 5'-AGTGATAGTGA-3' with K(a)=9.7 x 10(9) M(-1). Tandem 1b also bind the 11 bp site but with lower affinity affording a 15-fold specificity for the shorter binding site. Replacing a methylene group in the amino acid linker with an oxygen atom to form tandem polyamide ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-E](HN)gamma-PyPyPy-beta-Dp (4) where E represents the ether linker, resulted in that an 80-fold specificity for the 10 bp binding site over the 11 bp site.     

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.