Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins

The DNA sequence of bovine microsomal cytochrome b₅ has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome b_s in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b₅ cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D ¹HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b₅. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (T_m of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (T_m 78°C) reflected in the values of H_m, S_mand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome b_s is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

For the first time the pro-form of a recombinant cysteine proteinasehas been expressed at a high level in Escherichia coli. Thisinactive precursor can subsequently be processed to yield activeenzyme. Sufficient protein can be produced using this systemfor X-ray crystallographic structure studies of engineered proteinases.A cDNA clone encoding propapain, a precursor of the papaya proteinase,papain, was expressed in E.coli using a T7 polymerase expressionsystem. Insoluble recombinant protein was solubilized in 6 Mguanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6.A protein-glutathione mixed disulphide was formed by dilutioninto oxidized glutathione and 6 M GuHCl, also at pH 8.6. Finalrefolding and disulphide bond formation was induced by dilutioninto 3 mM cysteine at pH 8.6. Renatured propapain was processedto active papain at pH 4.0 in the presence of excess cysteine.Final processing could be inhibited by the specific cysteineproteinase inhibitors E64 and leupeptin, but not by pepstatin,PMSF or EDTA. This indicates that final processing was due toa cysteine proteinase and suggests that an autocatalytic eventis required for papain maturation.     

Molecular dynamics simulation of winter flounder antifreeze protein variants in solution: correlation between side chain spacing and ice lattice

The solution structure of the 38 amino acid C-terminal regionof the precursor for the HPLC-6 antifreeze protein from winterflounder has been investigated with molecular dynamics usingthe AMBER software. The simulation for the peptide in aqueoussolution was carried out at a constant temperature of 0°Cand at atmospheric pressure. The simulation covered 120 ps andthe results were analyzed based on data sampled upon reachinga stable equilibrium phase. Information has been obtained onthe quality of constant temperature and pressure simulations,the solution structure and dynamics, the hydrogen bonding network,the helix-stabilizing role of terminal charges and the interactionwith the surrounding water molecules. The Lys18–Glu22interactions and the terminal charged residues are found tostabilize a helical structure with the side chains of Thr2,Thr13, Thr24 and Thr35 equally spaced on one side of the helix.The spacing between oxygen atoms in the hydroxyl group of thethreonine side chains exhibits fluctuations of the order of2–3 Å during the 120 ps of simulation, but valuessimultaneously close to the repeat distance of 16.6 Åbetween oxygen atoms along the [0112] direction in ice are observed.Furthermore, two engineered variants were studied using thesame simulation protocol.     

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines

Using the well-characterized antibody McPC603 as a model, wehad found that the F_v fragment can be isolated from Escherichiacoli as a functional protein in good yields, whereas the amountof the correctly folded F_ab fragment of the same antibody producedunder identical conditions is significantly lower. In this paper,we analyse the reasons for this difference. We found that avariety of signal sequences function in the secretion of theisolated chains of the F_ab fragment or in the co-secretion ofboth chains in E.coli. The low yield of functional F_ab fragmentis not caused by inefficient expression or secretion in E.coli,but by inefficient folding and/or assembly in the periplasm.We compared the folding yields for the F_v and the F_ab fragmentin the periplasm under various conditions. Several diagnosticframework variants were constructed and their folding yieldsmeasured. The results show that substitutions affecting cis-prolineresidues and those affecting various disulphide bonds in theprotein are by themselves insufficient to dramatically changethe partitioning of the folding pathway to the native structure,and the cause must lie in a facile aggregation of folding intermediatescommon to all structural variants. However, all structural variantscould be obtained in native form, demonstrating the generalutility of the secretory expression strategy.     

A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.     

Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli

We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor.     

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

Cloning and expression in E.coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor X_a, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor X_a to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli.     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

Engineered turns of a recombinant antibody improve its in vivo folding

Using recombinant antibodies functionally expressed by secretionto the periplasm in Escherichia coli as a model system, we identifiedmutations located in turns of the protein which reduce the formationof aggregates during in vivo folding or which influence cellstability during expression. Unexpectedly, the two effects arebased on different mutations and could be separated, but bothmutations act synergistically in vivo. Neither mutation increasesthe thermodynamic stability in vitro. However, the in vivo foldingmutation correlates with the yield of oxidative folding in vitro,which is limited by the side reaction of aggregation. The invivo folding data also correlate with the rate and activationentropy of thermally induced aggregation. This analysis showsthat it is possible to engineer improved frameworks for semi-syntheticantibody libraries which may be important in maintaining librarydiversity. Moreover, limitations in recombinant protein expressioncan be overcome by single amino acid substitutions.     

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions

Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'–OmpA(46–159)–Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46–159), a second OmpA segment, consisting ofamino acids 46–66, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 46–84, 46–109, 46–128, 46–141and 46–145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Probing conserved amino acids in phospholipase D (Brassica oleracea var. capitata) for their importance in hydrolysis and transphosphatidylation activity

In addition to hydrolysis of glycerophospholipids, phospholipases D (PLDs) catalyze the head group exchange. The molecular basis of this transphosphatidylation potential, which strongly varies for PLDs from different sources, is unknown hitherto. Recently, the genes of two PLD isoenzymes from white cabbage have been sequenced and expressed in Escherchia coli, yielding the basis for mutational studies. In the present paper, three sequence characteristics of the isoenzyme (PLD2) that corresponds to the often used enzyme isolated from cabbage leaves have been probed for their importance in hydrolysis as well as transphosphatidylation activities: (i) the two HKD motifs, (ii) the C terminus and (iii) the eight cysteine residues. All these regions or amino acids are highly conserved in alpha-type plant PLDs. Based on multiple alignments, predictions of secondary structure and comparisons of hydrophobicity profiles, 35 enzyme variants were created and assayed. All positions tested proved to be very sensitive towards amino acid exchanges with respect to hydrolytic activity in the absence of glycerol as well as to the ratio of hydrolytic and transphosphatidylation activities in the presence of glycerol. A significant increase of total activity and transphosphatidylation activity could be obtained by the substitutions C310S and C625S.