C-terminus engineering of soybean proglycinin: improvement of emulsifying properties

Introduction of the extension region of beta-conglycinin alpha' subunit at the C-terminus of proglycinin A1aB1b results in the improvement of its emulsifying properties. To understand the basic for such improvement, we introduced the alpha' and alpha extension regions to the A2B1a C-terminus, and the alpha extension and A5A4B3 hypervariable regions, and an oligopeptide composed of 20 negatively or positively charged residues to the A1aB1b C-terminus, creating A2B1aalpha', A2B1aalpha, and A1aB1balpha, A1aB1bA4IV, A1aB1bNeg and A1aB1bPos, respectively. All the modified versions were produced in Escherichia coli. Their molecular size, thermal stability, surface hydrophobicity, solubility and emulsifying ability were studied. Analyses of molecular size and thermal stability suggested that all the modified versions formed the proper conformation similar to that of the wild type (WT). Solubility was intrinsic to each mutant. At ionic strength 0.5, the emulsifying abilities of all mutants were better than that of the WT except A1aB1bPos and A1aB1bNeg, and at ionic strength 0.08, all mutants especially A1aB1bPos exhibited better emulsifying ability than did the WT. The order of stability of the emulsion at both ionic strengths (0.08 and 0.5) was A1aB1balpha >or= A2B1aalpha > A1aB1balpha' >or= A2B1aalpha' > A1aB1bPos > A1aB1bA4IV >or= A1aB1bNeg > A1aB1b, A2B1a. These results indicate that the emulsion stability of proglycinin mutants depends on length and hydropathy profile of the polypeptides added to the C-terminus of proglycinin.     

Thermal densturation of glycinin as a function of hydration

Hydration effects on the thermal stability of glycinin (soybean 11S protein) were examined by differential scanning calorimetry (DSC). In a model system with pure glycinin, the denaturation temperature (T_d) decreased with increasing moisture. Between 22 and 44% moisture, two endotherms were observed, where the lower-temperature endotherm became progressively reduced in magnitude with a concomitant increase in a higher-temperature transition. At 45.5% moisture, only a single endotherm was observed. The regression curves over the entire moisture range from 2 to 66% were derived as asymptote functions, where M equals the percentage total moisture. Equations were developed from the curves, and the relationship between T_d and moisture were: T_d=92.4+196.5e^−0.068M and, for the low-temperature endotherm, 82.4+144.3e^−0.068M. By interaction of 11S protein with either ethanol, a neutral detergent (Triton X-100) or 40% sucrose, both one- and two-endotherm curves were generated. Such calorimetric behavior is indicative of nonequilibrium denaturation and supports the notion that structure reorganization during DSC is water content-dependent. Our findings suggest that either glycinin’s acidic/basic subunits or a change in secondary protein structure may give rise to two endotherms. Presented at the 1993 AOCS Meeting, April 25–29, 1993, Anaheim, CA.     

MTG酶诱导大豆11S球蛋白透明冷致水凝胶的制备

水凝胶的制备逐步朝向安全无毒、高生物相容性的方向发展。本工作着重探究一种大豆球蛋白透明冷致水凝胶的制备方法。首先利用葡聚糖硫酸盐（dextran sulphate, DS）与大豆11S球蛋白（glycinin, 11S）在加热过程中形成多阴离子复合物,继而采用微生物转谷氨酰胺酶（microbial transglutaminase, MTG酶）在pH 7.0、一定离子强度条件下,通过分子间ε-（γ-谷氨酰胺基）赖氨酸共价键交联,制备了透明冷致水凝胶,并对其流变学、质构特性、持水性及微结构进行研究。结果表明：葡聚糖硫酸盐（DS）的添加赋予大豆11S球蛋白溶液较高的荷电量,在中性条件下,MTG酶具有交联此高荷电多阴离子复合物的能力,从而在较低温度下形成透明冷致水凝胶。通过控制相似文献   

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.     

Functional properties of the acidic and basic subunits of the glycinin (11S) soy protein fraction

The functional properties of low- and high-M.W. (LMW and HMW, respectively) acidic subunits and the basic subunit separated from the 11S soy protein fraction were studied and compared with the functional properties of the 11S fraction. Among the functional properties investigated were solubility, emulsification, and viscosity. The results showed that the LMW acidic subunit had higher solubility than the HMW acidic subunit. Among all the samples, the LMW subunit separated by using β-mercaptoethanol (ME) was the most soluble, with a solubility of 98–100% at a pH of 6–12. The solubility profile of the HMW subunit followed a pattern similar to the solubility of 11S. The lowest solubility was observed around pH values in the range close to the isoelectric point for both the LMW and HMW subunit. The basic subunit was not soluble in the pH range 3–10; however, the solubility increased more than 50% at pH 13 compared to the solubility at pH 10. The emulsification capacity of all subunits was higher than 11S in the following descending order: LMW, basic, HMW, 11S. Emulsification activity and stability of the subunits were greater than those of the 11S samples at room temperature and 95°C. With the exception of the LMW subunit separated with ME, the subunits had a higher viscosity than 11S. The basic subunit separated with sodium bisulfite had the highest viscosity of all the samples tested.     

The influence of genotypic variation in protein composition on emulsifying properties of soy proteins

This study describes the relationship between the emulsifying properties of soybean proteins and their composition, i.e., glycinin (11S) and β-conglycinin (7S). Twelve investigated soybean genotypes showed significant differences in storage protein composition. The β-conglycinin concentration positively correlated with extractable soluble protein content, which was positively correlated with protein extractability. These data suggest that the level of β-conglycinin has a positive influence on protein extractability. The emulsion activity index (EAI) was strongly and positively correlated with the 11S∶7S ratio and strongly and negatively correlated with the concentration of β-conglycinin. The emulsion stability index (ESI) showed a moderate positive correlation with the monomeric form of glycinin and a strong positive correlation with the ratio of the monomeric to dimeric form of glycinin. No association was evident between ESI and EAI. Also, no relationship was found between ESI or EAI and extractability. Based on these data, it appears that the 11S∶7S ratio strongly reflects the ability of soybean proteins to form emulsions, whereas the ratio of the two different forms of glycinin may be crucial factors for the stability of soybean protein emulsions. Thus, understanding the relationship between protein composition and functionality could be useful for further improvement of functional behavior of soy proteins in food systems.     

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants

A microbial expression system for the study of the cysteineprotease papain has been developed as a more useful alternativeto the insect cell/baculovirus expression system we have previouslyused. A synthetic papain precursor (propapain) gene was expressedin the yeast Saccharomyces cerevisiae under the control of the-factor promoter. Efficient expression required fusion of thepropapain sequence with the yeast -factor prepro region anda yeast host cell defective in the synthesis of vacuolar proteases.Surprisingly, the glycosylated form of the inactive papain precursoris not secreted, but accumulates within the yeast cell. Completeconversion of the intracellular zymogen into active mature papaincould be achieved in vitro. Purified recombinant papain producedby the yeast system has kinetic characteristics similar to thoseof the natural enzyme. An advantage of the yeast expressionsystem over the baculovirus/insect cell system is that we canperform mutagenesis and screening of papain mutants very efficiently.We have set up a ‘one-tube’ screening procedurefor the simultaneous characterization of numerous mutants ofthe papain precursor. Yeast cells are grown and lysed in microtiterplate wells and the released papain precursor is then activatedto mature papain. This assay allows easy discrimination betweenproteins with close to wild type properties and proteins thatare not functional. We have applied this assay to investigatethe spectrum of amino acids which are tolerated at Asnl75 ofpapain using two independently derived libraries of mutantsat this position. Many amino acid substitutions at this positionare not accepted; only the reintroduction of Asn restored normalfunction.     

Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

The feasibility of stabilizing proteins towards proteolyticdegradation was explored by engineering the primary proteolyticcleavage site(s). This novel approach does not require informationon the 3-D structure of the native enzyme. As a model system,the extracellular lipase of Pseudomonas glumae was chosen, whichis sensitive towards degradation by subtilisin-tvpe proteases.The primary proteolytic cleavage in the lipase appeared to belocated between amino acids serine 153 and histidine 154. SincesubtUisins are known to show a preference towards amino acidresidues surrounding the scissile bond, non-preferred aminoadds were introduced in this area. Two concepts were tested:the introduction of arginine or glutainate residues (chargeconcept) and the introduction of proline residues (proUne concept).Although the mutant Upases produced according to either of theseconcepts were still cleaved in the same area, they showed aconsiderably increased stability towards proteolytic degradation.     

The creation of a novel fluorescent protein by guided consensus engineering

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

Functional properties of soybean and lupin protein concentrates produced by ultrafiltration-diafiltration

Ultrafiltration followed by diafiltration (UF-DF) was evaluated for the production of protein products from partially defatted soybean meal or undefatted lupin (Lupinus albus L.2043N) meal. This study determined the effects of UF-DF on functional properties of the extracted proteins and compared the results with those of protein prepared by acid-precipitation (AP). UF-DF produced only protein concentrates (73% crude protein, dry basis, db), while AP produced protein isolates (about 90% crude protein, db). Soybean protein produced by UF-DF showed markedly higher values for solubilities up to pH 7.0, surface hydrophobicity index, emulsion activity index, and foaming capacity than did the AP soybean protein. UF-DF soy protein was also the most heat-stable among all protein samples tested. With lupin proteins, only the surface hydrophobicity and emulsion activity indices were significantly improved by using UF-DF. UF-DF generally had no adverse effects on, and in most cases even improved, the functional properties of soy protein concentrate produced by this method. UF-DF did not produce a comparable improvement in functional properties of lupin proteins as it did for soybean protein.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

A monomeric variant of triosephosphate isomerase (TIM) witha new engineered binding groove has been characterized further.In this variant (ml8bTIM), the phosphate binding loop had beenshortened, causing the binding site to be much more extended.Here, it is reported that in the V233A variant of ml8bTIM (A-TIM),three important properties of the wild-type TIM active sitehave been restored: (i) the structural properties of loop-7,(ii) the binding site of a conserved water molecule betweenloop-7 and loop-8 and (iii) the binding site of the phosphatemoiety. It is shown that the active site of A-TIM can bind TIMtransition state analogs and suicide inhibitors competently.It is found that the active site geometry of the A-TIM complexesis less compact and more solvent exposed, as in wild-type TIM.This correlates with the observation that the catalytic efficiencyof A-TIM for interconverting the TIM substrates is too low tobe detected. It is also shown that the A-TIM active site canbind compounds which do not bind to wild-type TIM and whichare completely different from the normal TIM substrate, likea citrate molecule. The binding of this citrate molecule isstabilized by hydrogen bonding interactions with the new bindinggroove.     

Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding

A gene encoding a bacterial IgG Fc binding domain was designedand synthesized. The synthetic DNA fragment was cloned 3' toan inducible trpE promoter such that expression of the genein Escherichia coli produced abundant Fc binding protein fusedto the first seven amino acids of the trpE protein. The recombinantprotein contained a single Fc binding domain and demonstratedefficient binding to'human IgG in Western blot analysis. Thisprotein degraded rapidly following cell lysis in the absenceof protease inhibitors, but could be effectively protected bythe addition of protease inhibitor. After purification of theprotein by IgG affinity chromatography, IgG Fc binding abilitywas retained for at least 24 h at either 23 or 37°C andon heating for 15 min at temperatures up to 65°C. No immunoprecipitationwas observed in interactions between the monodomain Fc bindingprotein and IgG molecules. Unlike staphylococcal protein A,no detectable binding of the monodomain IgG Fc binding proteinwas observed to either IgM or IgA. Truncated proteins, expressedfrom a series of 3' deletions of the synthetic gene, were usedto estimate the minimum portion of a monodomain Fc binding proteinthat retained Fc binding ability.     

Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Databaseof tertiary architectures that displayed oligomeric association(24 molecules) or whose polypeptide folding revealed domains(34 proteins). The subunit and domain interfaces for these proteinswere respectively examined from the following aspects: percentagewater-accessible surface area buried by the respective associations,surface compositions and physical characteristics of the residuesinvolved in the subunit and domain contacts, secondary structuralstate of the interface amino acids, preferred polar and non-polarinteractions, spatial distribution of polar and non-polar residueson the interface surface, same residue interactions in the oligomeric:contacts, and overall cross-section and shape of the contactsurfaces. A general, consistent picture emerged for both thedomain and subunit interfaces.     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1.     

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus

A novel protein able to bind with high affinity to the Fc fragmentof IgG from a variety of animals has been produced by a genesynthesis approach. The IgG binding is accomplished by the presenceof a single or two consecutive domains based upon domain B fromprotein A of Staphylo-coccus aureus. The IgG-binding moietyis fused to a peptide containing 21, 53 or 81 amino acids derivedfrom the N-terminus of bovine DNase I. The latter is presentto guide the expression of the protein in Escherichia coli intoan inclusion body. This facilitates the high expression andrecovery of the IgG-binding domains. The binding activity ofthis fusion protein is very close to that of the native proteinA. Site-directed mutagenesis of the fusion protein and subsequentidentification of changed binding interactions is reported.     

Protein fragment variability analysis and some principles of protein engineering of vaccines

Based on protein sequence databank (PIR), the ‘variablefragment’ bank, comprising pairs of closely-related proteins,containing one or more strongly differing sites of primary structures,was formed. The bank includes 465 ‘variable fragments’in 383 protein pairs. The amino acid composition of ‘variablefragments’ was examined and indices of potential aminoacid residue variability were formed. An analysis of the interchangeabilityof amino acid fragments depending on the substitution site (N-or C-terminal, or middle part of a chain), the fragment lengthdifferences and physico-chemical properties of residues, suchas volume, hydrophobicity, polarity and isoelectric point, wascarried out. Based on this analysis some general empirical rulesof peptide insertions in carrier proteins were created. Therules are directed at performing modifications leaving the generalstructure and function of the carrier protein molecule unchanged.The selection scheme for the regions suitable for modificationand the criteria for determination of the range of acceptablevariations in these regions were suggested. The use of the potentialvariability profile for detecting regions suitable for peptideinsertion was considered using surface protein of hepatitisB virus as an example.     

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.