Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

This paper reports the expression of an artificial functionalpolypeptide in bacteria. The gene of a designed 24-residue DDT-bindingpolypeptide (DBP) was inserted between the BamHI and PstI cleavagesites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, wascloned in Escherichia coli JM109. After induction by isopropyl-ß-D-thiogalactopyranosidea fusion protein was expressed in which DBP was linked to theCOOH-termiuus of ß-galactosidase. DBP, which is stableto trypsin, was obtained by tryptic digestion of the fusionprotein and subsequent fractionation of the tryptic peptidesby reversed-phase h.p.l.c. Recombinant and chemically synthesizedDBP showed identical chromatographic properties, amino acidcomposition, and chymotryptic digestion patterns. Both the ß-galactosidase-DBPfusion and isolated recombinant DBP bound DDT. The fusion proteinwas 25 times as potent as the designed 24-residue DBP in activatinga cytochrome P-450 model system using equimolar catalytic amountsof the two proteins.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active siteof Escherichia coli alkaline phosphatase was investigated bysite-specific mutagenesis. A mutant version of alkaline phosphatasewas constructed with alanine in place of aspartic acid at position101. When kinetic measurements are carried out in the presenceof a phosphate acceptor, 1.0 M Tris, pH 8.0, both the k_cat andthe K_m, for the mutant enzyme increase by –2-fold, resultingin almost no change in the k_cat/K_m ratio. Under conditions ofno external phosphate acceptor and pH 8.0, both the k_cat andthe K_m for the mutant enzyme decrease by {small tilde}2-fold,again resulting in almost no change in the k_cat/K_m ratio. Thek_cat for the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-typeand mutant enzymes, as is the K₁ for inorganic phosphate. Thereplacement of aspartic acid 101 by alanine does have a significanteffect on the activity of the enzyme as a function of pH, especiallyin the presence of a phosphate acceptor. At pH 9.4 the mutantenzyme exhibits 3-fold higher activity than the wild-type. Themutant enzyme also exhibits a substantial decrease in thermalstability: it is half inactivated by treatment at 49°C for15 min compared to 71°C for the wild-type enzyme. The datareported here suggest that this amino acid substitution altersthe rates of steps after the formation of the phospho-enzymeintermediate. Analysis of the X-ray structure of the wild-typeenzyme indicates that the increase in catalytic rate of themutant enzyme in the presence of a phosphate acceptor may bedue to an increase in accessibility of the active site nearSerl02. The increased catalytic rate of this mutant enzyme maybe utilized to improve diagnostic tests that require alkalinephosphatase, and the reduced heat stability of the mutant enzymemay make it useful in recombinant DNA techniques that requirethe ability to heat-inactivate the enzyme after use.     

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design

The results of a protein design project are used to comparedifferent predictive strategies with respect to proteinproteininteractions. We have been able to generate variants of humanpancreatic secretory trypsin inhibitor (hPSTI) optimized withrespect to the affinity and specificity for human leukocyteelastase relative to trypsin and chymotrypsin, and in particularchymotrypsin. The extremely strong and specific human leukocyteelastase inhibitors were thus developed in three rounds of mutagenesisand two rounds of 3-D modelling; only 24 variants in total weresynthesized, although variations at seven different amino acidpositions were involved (i.e. from 20⁷ possible variants). Anexcellent elastase inhibitor could be designed with the minimumof two amino acid exchanges. The value of structural modellingand actual structure determination is discussed in the lightof the experimental results of the designed protein variantsand the results of tertiary structure determinations of thefree variant and the inhibitorprotease complex. Particular referenceis given to the strategy to be followed in protein design projectsin general and to the development of protease inhibitors inparticular.     

Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity

We developed a novel bivalent antibody fragment, the linear(L-) F(ab')₂, comprising tandem repeats of a heavy chain fragmentV_H–C_H1–V_H–C_H1 cosecreted with a light chain.Functional humanized L-F(ab')₂ directed against p185^HER2 wassecreted from Escherichia coli at high titer (100 mg/l) andpurified to homogeneity. The L-F(ab')₂ binds two equivalentsof antigen with an apparent affinity (K_d = 0.46 nM) that iswithin 3-fold of the corresponding thioether-linked F(ab')₂fragment The N-terminal site binds antigen with an affinity(K_d = 1.2 nM) that is 4-fold greater than that for the C-terminalsite, as shown by the comparison of L-F(ab')₂ variants containinga single functional binding site. L-F(ab')₂ has greater antiproliferativeactivity than the thioether-linked F(ab')₂ against the p185^HER2-overexpressingtumor cell line BT474. Linear and thioether-linked F(ab')₂ havevery similar pharmacokinetic properties in normal mice, andtheir serum permanence times are respectively 7- and 8-foldlonger than the corresponding Fab fragment L-F(ab')₂ offersa facile route to bivalent antibody fragments that are potentiallysuitable for clinical applications, and that may have improvedbiological activity compared with thioether-linked F(ab')₂ fragments.     

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

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     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli

Mutants of colicin A have been prepared in which the three tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.     

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC11105 was cloned into pUC 9 and the resulting vector (pUPA-9),when transformed into E.coli strain 5K, allowed the constitutiveoverproduction of mature penicillin acylase when grown at 28°C.The enzyme ws purified from the periplasmic fraction of E.colipUPA-9 by hydrophobic interaction chromatography and anion exchange.Crystals of penicillin acylase were grown in batch using polyethyleneglycol 8000 as a precipitant. The crystals (space group P1)diffracted to beyond 2.3 Å.     

Crystal structure of T4-lysozyme generated from synthetic coding DNA expressed in Escherichia coli

The polypeptide produced by expressing a chemically synthesizedgene coding for the amino-acid sequence of T4-lysozyme has beencrystallized and subjected to X-ray diffraction. The crystalstructure has been refined to a standard R-factor of 0.191 fordata between 8 and 2 ? resolution. The refined model is essentiallythe same as the well-known structure of wild-type T4-lysozymedetermined previously by Matthews et al. (1987). Some smallchanges in the C-terminal region, which is important in maintainingthe folded structure, have been noted. In addition to confirmingthat the synthetic gene product is very close to the wild type,this structure provides a benchmark for protein engineeringexperiments on the folding and the catalytic activity of thismolecule by the method of gene synthesis.     

Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli

An advantage of exporting a recombinant protein to the periplasmof Escherichia coli is decreased proteolysis in the periplasmcompared with that in the cytoplasm. However, protein degradationin the periplasm also occurs. It has been widely accepted thatthe thermodynamic stability of a protein is an important factorfor protein degradation in the cytoplasm of E.coli. To investigatethe effect of the thermodynamic stability of an exported proteinon the extent of proteolysis in the periplasm, barnase (an extracellularribonuclease from Bacillus amyloliquefaciens) fused to alkalinephosphatase leader peptide was used as a model protein. A setof singly or doubly mutated barnase variants were constructedfor export to the E.coli periplasm. It was found that the half-lifeof the barnase variants in vivo increased with their thermodynamicstability in vitro. A dominant factor for the final yield ofexported barnase was not exportability but the turnover rateof the barnase variant. The yield of a stabilized mutant wasup to 50% higher than that of the wild type. This suggests thatexporting a protein to the periplasm and using protein engineeringto enhance the stability can be combined as a strategy to optimizethe production of recombinant proteins.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin.     

Synthetic genes for human muscle-type adenylate kinase in Escherichia coli

An artificial gene coding for the human muscle-type cytosolicadenylate kinase (hAK1) was chemically synthesized and directlyexpressed in Escherichia coli under the control of trp promoter.The DNA duplex of 596 bp was designed and constructed from 40oligonucleotide fragments of typically 30 nucleotides in length.Twelve unique restriction sites were fairly evenly spaced inthe synthetic gene to facilitate site-specific mutagenesis atany part of this recombinant protein. The genes for mutant hAK1(Tyr 95 – Phe 95, Y95F hAK1; Arg 97 – Ala 97, R97AhAK1) were constructed by cassette mutagenesis and utilizedrestriction sites incorporated in the hAK1 gene. The recombinanthAK1 was purified to homogeneity by a two-step chromatographicprocedure with a good yield, and showed the same adenylate kinaseactivity as that of authentic hAK1. preliminary kinetic studiesshow that the enzymatic activity (V_{max app,cor})     

Generation and analysis of proline mutants in protein G

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1–301, and the synthetase(Syn) domain as residues 304–935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292–310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme.     

The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E.coli

Two refined crystal structures of aspartate aminotransferasefrom E.coli are reported. The wild type enzyme is in the pyridoxalphosphate (PLP) form and its structure has been determined to2.4 Å resolution, refined to an R-factor of 23.2%. Thestructure of the Arg292Asp mutant has been determined at 2.8Å resolution, refined to an R–factor of 20.3%. Thewild type and mutant crystals are isomorphous and the two structuresare very similar, with only minor changes in positions of importantactive site residues. As residue Arg292 is primarily responsiblefor the substrate charge specificity in the wild type enzyme,the mutant containing a charge reversal at this position mightbe expected to catalyze transamination of arginine as efficientlyas the wild type enzyme effects transamination of aspartate[Cronin,C.N. and Kirsch,J.F. (1988) Biochemistry, 27, 4572–4579].This mutant does in fact prefer arginine over aspartate as asubstrate, however, the rate of catalysis is much slower thanthat of the wild type enzyme with its physiological substrate,aspartate. A comparison of these two structures indicates thatthe poorer catalytic efficiency of R292D, when presented witharginine, is not due to a gross conformational difference, butis rather a consequence of both small side chain and main chainreorientations and the pre–existing active site polarenvironment, which greatly favors the wild type ion pair interaction.     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli

Bacterial expression systems can greatly facilitate proteinengineering of antibodies. We have developed a system for high-levelexpression of antibodies, antibody fragments, or hybrid antibodieswith novel effector functions in the periplasm of Escherichiacoli. From 5 ml of cells, a simple extraction yields sufficientmaterial for SDS-gel electro-phoresis, detection and characterizationof hapten binding. To demonstrate our system, heavy-chain variableregions and 1 light chains of a mouse anti-NP antibody weresynthesized as hybrid proteins with a bacterial signal peptide(Omp F). Each chain is secreted into the periplasm where processing(cleavage of the signal peptide), folding and heterodimer associationtake place. Periplasmic proteins are released by cold osmoticshock, and hapten-binding activity is easily detected withoutfurther manipulation. The ease of genetic engineering in thissystem will facilitate the production of immunoglobulin derivativesdesigned for specific applications, and expression of thesemolecules in a native state will allow the rapid screening ofcombinatorial libraries and the results of mutagenesis.