Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

Directed evolution of proteins for increased stability and expression using yeast display

The expression of recombinant proteins incorporated into the cell wall of Saccharomyces cerevisiae (yeast surface display) is an important tool for protein engineering and library screening applications. In this review, we discuss the state-of-the-art yeast display techniques used for stability engineering of proteins including antibody fragments and immunoglobulin-like molecules. The paper discusses assets and drawbacks of stability engineering using the correlation between expression density on the yeast surface and thermal stability with respect to the quality control system in yeast. Additionally, strategies based on heat incubation of surface displayed protein libraries for selection of stabilized variants are reported including a recently developed method that allows stabilization of proteins of already high intrinsic thermal stability like IgG1-Fc.     

Rapid method for measuring ScFv thermal stability by yeast surface display

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.     

Chemical modification of silk sericin in lithium chloride/dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate

This paper reports chemical modification of silk sericin in LiCl/dimethyl sulfoxide (DMSO) solvent with 4-cyanophenyl isocyanate. Sericin is a highly hydrophilic protein secreted by Bombyx mori, serving as a protein glue in a cocoon. LiCl/DMSO was found to be a good solvent of sericin and useful for homogeneous modification of its abundant hydroxyl groups under nonaqueous condition. Fourier transform infrared (FTIR) analysis of the modified sericins revealed that 4-cyanophenyl groups were incorporated into sericin molecules mainly through urethane linkages. Several characteristics of the modified sericins such as solubility characteristic, hygroscopic property, and thermal stability were investigated. Secondary structure analysis using FTIR spectra suggested that formation of strong intermolecular hydrogen bonds was inhibited by the modification that is probably attributable to the incorporation of bulky 4-cyanophenyl groups. These results demonstrate that chemical modification of sericin using LiCl/DMSO solvent markedly alters its characteristics.     

豆壳过氧化物酶的盐酸胍变性与化学修饰研究

研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。     

The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density

Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. (31)P NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.     

Effect of chemical modification on thermal stability of horseradish peroxidase]

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.     

High-level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene

This study was designed to produce yeast (Schwanniomyces occidentalis) phytase in rice with a view to future applications in the animal feed industry. To achieve high-level production, chimeric genes with the secretory signal sequence of the rice chitinase-3 gene were constructed using either the original full-length or N-truncated yeast phytase gene, or a modified gene whose codon usage was changed to be more similar to that of rice, and then introduced into rice (Oryza sativa L.). When the original phytase genes were used, the phytase activity in the leaves of transgenic rice was of the same level as in wild-type plants, whose mean value was 0.039 U/g fresh weight (g-FW) (1 U of activity was defined as 1 micromol P released per min at 37 degrees C). In contrast, the enzyme activity was increased markedly when codon-modified phytase genes were introduced: up to 4.6 U/g-FW of leaves for full-length codon-modified phytase, and 10.6 U/g-FW for truncated codon-modified phytase. A decrease in the optimum temperature and thermal stability was observed in the truncated heterologous enzyme, suggesting that the N-terminal region plays an important role in enzymatic properties. In contrast, the optimum temperature and pH of full-length heterologous phytase were indistinguishable from those of the benchmark yeast phytase, although the heterologous enzyme was less glycosylated. Full-length heterologous phytase in leaf extract showed extreme stability. These results indicate that codon modification, combined with the use of a secretory signal sequence, can be used to produce substantial amounts of yeast phytase, and possibly any phytases from various organisms, in an active and stable form.     

酵母乙醇耐受性状遗传不稳定的表观遗传探讨

诱变、驯化等传统手段获得理想性状的酵母菌株,其性能在传代和保存过程中很容易发生性状丢失现象。从表观遗传学的角度出发,初步探讨酵母菌株在传统的诱变、驯化等育种过程及其性状丢失的表观遗传的分子机理。采用驯化等手段选育耐乙醇酵母,并通过无压力方式传代,研究此过程中酵母乙醇耐受性状遗传的稳定性与耐受相关的pro1、tps1、sod1基因启动子区域结合组蛋白上H3K4甲基化水平的关系。结果表明酵母乙醇耐受性状的变化受到酵母表观遗传控制。控制表观遗传的修饰过程易受环境改变的影响,因此经过选育获得的乙醇耐性性状遗传的不稳定性可能与表观遗传分子机理密切相关。     

Soluble high molecular weight derivatives of pancreatic trypsin inhibitor. Effect of electrostatic interaction of protein with the carrier on covalent binding and some physico-chemical properties of modified preparations of pancreatic inhibitor

Modification of pancreatic trypsin inhibitor by carboxymethyl dextran with the aid of carboxyl groups of the carrier activated by water-soluble carbodiimide has been carried out. A special role of electrostatic interactions of protein with the carrier during inhibitor modification has been shown. This modification results in active preparations with high protein concentration on the carrier; their molecular weight is practically equal to that of previously used carrier. A comparison of the thermal stability of high molecular weight preparations of pancreatic inhibitor with respect to protein concentration on the carrier and the number of links between protein and carrier has been carried out.     

Preparation and characterization of novel optically active poly(vinyl alcohol-co-vinyl ester) in nonaqueous medium using <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine as a chiral material

In this investigation, poly(vinyl alcohol) was chemically modified by the introduction of different amounts of N-phthaloyl-l-phenylalanine. The modification was carried out by the reaction of PVA hydroxyl groups with (2S)-3-phenyl-2-phthalimidylpropanoyl chloride using N,N-dimethyl acetamide/lithium chloride as a reaction media. The novel copolymers obtained were characterized by spectroscopic techniques, elemental analysis, X-ray diffraction and thermal methods. Optical rotation and viscosities were also measured. The degree of esterification was determined by ¹H-NMR. The influence of reagent molar ratio on the degree of modification was also evaluated. The vinyl(3-phenyl-2-phthalimidopropanoate) content in the copolymer was attained up to 52%. Thermal stability of the copolymers was checked by thermogravimetric analysis and differential thermogravimetric analysis. All copolymers displayed improved thermal stability compared to the parent polymer.     

Modification of porcine pancreatic lipase with Z-proline

Porcine pancreatic lipase was modified with Z-proline via the constitution of amide bonds between the free amino groups of lipase and the carboxyl groups of Z-proline, which were activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Different amounts of Z-proline were bound to lipase. Modification degree was determined by 2,4,6-trinitrobenzene sulphonic acid (TNBS), by means of the decrease in free amino groups on lipase. The reason for choosing Z-proline was its unique structural characteristics, protected amino groups, and its effect on protein conformation by reducing the flexibility of the lipase molecule, thus achieving stabilization against changes in pH and temperature.After the modification, porcine pancreatic lipase was found to have new physicochemical characteristics, such as optimum alkaline pH stability and thermal stability at elevated temperatures.     

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.     

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.     

Permeability properties of chemically modified porin trimers from Escherichia coli B

The pore-forming protein of the outer membrane of Escherichia coli, porin, was chemically modified with acetic anhydride, succinic anhydride, and glycinamide. Extensive modification of amino groups of the functional porin trimers caused reduced diffusion rates of the negatively charged solutes such as p-nitrophenyl phosphate and AMP, but did not reduce significantly the diffusion of positively charged molecules carbobenzoxy-glycyl-prolyl-arginine-p-nitranilide and tosyl-glycyl-prolyl-arginine-p-nitranilide. Modification of carboxyl groups of trimers caused decreased diffusion rates of the positively charged solutes more significantly than the diffusion rates of negatively charged solutes. The results suggest that the ionic interactions play an important role for the diffusion of charged solutes through the porin pore. The diffusion of p-nitrophenyl alpha-D-glucoside, an uncharged solute, ws not influenced significantly by modification of either amino or carboxyl groups. This observation suggests that modifications only occurred in areas outside of the narrowest portion of the pore or, alternatively, that amino and carboxyl groups are exclusively located at noncylindrical area of the pore. The structural integrity of the acetylated and the succinylated trimers seemed well preserved. On the other hand, modification of carboxyl groups decreased the thermal stability of trimers and extensive modifications caused the dissociation of trimers into monomers at 37 degrees C.     

Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A.

In order to probe changes in the structural stability induced by the introduction of hydrophobic groups into proteins, the amino groups of ribonuclease A and chymotrypsinogen A were reductively alkylated by reaction with various aliphatic aldehydes, formaldehyde, acetaldehyde, n-butylaldehyde and n-hexylaldehyde, and their thermal stabilities were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Ribonuclease A was thermally unstabilized by reductive alkylation, while chymotrypsinogen A was slightly stabilized, depending on both the size of the introduced alkyl groups and the extent of modification. These observations suggest that the effects induced by alkylation involve not only steric hindrance due to the entering bulky groups but also certain other factors such as the participation of the chemically introduced alkyl groups in hydrophobic interactions.     

The conformation properties and conformation stability of streptokinase and its polymer derivative

The properties and conformational stability of the proteinaceous activator of fibrinolysis--native streptokinase--and its derivative obtained by modification with a linear hydrophilic copolymer based on N-vinylpyrrolidone, were studied by the circular dichroism method. It was shown that polymeric modification of streptokinase had no effect on the secondary structure, while the conformational stability of the modified protein to urea was higher than that of the native one. Studies on thermal stability of both native and modified forms of streptokinase showed that the inactivation rate was lower in the modified form as compared to the native one.     

Physical property improvements of a pellicular biocatalyst

The thermal stability of an immobilization technique using a pellicular latex matrix was examined in a packed-bed column reactor. The stability was found to vary with liquid flow rate, the type of latex, temperature of operation, and the amount of yeast cells. Adjusting these parameters and introducing particulate inorganic fillers strengthened the latex matrix and improved the thermal stability. Optimization of this immobilization technique resulted in a procedure that allowed latex polymers to be mechanically stable at temperatures up to 50°C. The biological viability of this improved immobilization scheme was demonstrated through the production of L-aspartic acid by immobilized cells of E. coli.     

Effect of polyamines on yeast cell-free protein synthesizing system. II. Increase stability of cell-free system in the presence of spermine.

The addition of spermine, at concentration which stimulates protein synthesis, to the yeast cell-free system significantly increases the thermal stability of the latter. Similar stabilizing effect of polyamine is observed for ribosome-poly U-ac-phe-tRNA complexes. These results suggest that the stimulatory effect of polyamines on the in vitro protein synthesis might be partly due to the increased stability of ribosomes and ribosome-peptydyl-tRNA complexes.     

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T_m-values up to 85 °C was developed. Besides library construction by error prone PCR, strong heat stress at 79 °C for 10 min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.