Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein

The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding.     

A new expression system for protein crystallization using trimeric coiled-coil adaptors

We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils, using the corresponding GCN4 variants.     

A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

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})     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosusfimbrial subuits of different serotypes suggests a degree offlexibility, which might be exploited for protein engineeringapproaches for the expression of other peptides. We have testedthis using the well-characterized peptide epitope from VP1 offoot-and-mouth disease virus (FMDV), residues 144–159:LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage,several oligonucleotides were designed for the substitutionof this sequence internally at hypervariable regions of thefimbrial subunit (aligned for maximum homology), and for itsaddition at the carboxy-terminus with a diglycine spacer asa flexible hinge. Following site-directed mutagenesis in phageM13, the modified genes were placed under P_L promoter controland placed in a broad host range vector. Analysis of the variantproteins expressed in E.coli showed that these substitutionsaffected, to varying extents, recognition by both anti-fimbrialand anti-FMDV antibodies, indicating that hypervariable region2 is a major antigenic determinant of the fimbrial subunit andthat local stereochemical effects can influence antibody bindingto the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa,viable transformants could only be obtained with the mutantgene encoding the carboxy-terminal graft. These cells providedfimbrial preparations comprised of the modified subunit. Thisthen constitutes the prototype for the development of a generalexpression system for the production of vaccine epitopes andother bioactive peptides. Furthermore, there is considerablescope for further modification of the system, for example byengineering specific chemical or protease cleavage sites forrelease of the grafted peptide. Alternatively, the fimbriaethemselves may serve as a useful supramoleuclar carrier or adjuvantfor immune provocation.     

Negatively charged purification tags for selective anion-exchange recovery

A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.     

Recombinant rabbit uteroglobin expressed at high levels in E.coli forms stable dimers and binds progesterone

In order to express uteroglobin in Escherichia coli we haveconstructed a DNA coding for complete mature rabbit uteroglobinby fusing genomic sequences from the second exon of the geneto an incomplete cDNA. This DNA was inserted into various positionsof the polylinker cloning region of pDS expression vectors andthe uteroglobin gene was expressed in E.coli by IPTG induction.Four different uteroglobinderived proteins were produced containing1, 3,5 and 7 more N-terminal amino acids than the naturallyoccurring mature protein. The yield of soluble protein stronglyincreased with increasing length of the N-terminal additions.Protein and RNA analysis showed that this variation is mostlikely due to progressively higher translation efficienciesof the larger recombinants. UG7, the most efficiently synthesizedrecombinant protein, carrying seven additional N-terminal aminoacids, was purified and further characterized. Like naturaluteroglobin, UG7 forms a dimer and binds progesterone with anaffinity indistinguishable to the natural protein. This bacteriallyproduced protein can be used for detailed structure–functioninvestigations of uteroglobin.     

Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins

Using the techniques of genetic engineering and the principlesof protein de novo design, we have developed a unique affinitymatrix protein tag system as a rapid, convenient and sensitivemethod to detect, purify and characterize newly expressed recombinantpeptides or proteins from cell extracts. The method utilizestwo de novo-designed linear peptide sequences that can selectivelydimerize to form the stable protein motif, the two-stranded-helical coiled-coil. In this method, a recombinant bacterialexpression vector pRLDE has been engineered so that one of thedimerization strands (E-coil) is expressed as a C-terminal fusiontag on newly expressed peptides or proteins, while the other(K-coil) is either biotin-labeled for detection in a Westernblot-type format or immobilized on an insoluble silica supportfor selective dimerization affinity chromatography. Recombinantlyexpressed peptides from Escherichia coli containing the dimerizationtag have been produced, detected and purified using this method.The recombinant peptides were easily and clearly identifiedusing the biotin-labeled coil, while the single-step affinitypurification results indicated the purity of the affinity purifiedexpressed peptides to be >95%, as assessed by reversed-phasechromatography. The stability of the dimerization domain alsoallows for the purified peptide to be left attached to the matrix,thus creating a new peptide-bound column that can be used tostudy peptide–protein or peptide–ligand interactions.Therefore this system offers a new alternative to existing peptideor protein fusion tags and demonstrates the utility of a denovo-designed system.     

Insertion of a disulfide-containing neurotoxin into E.coil alkaline phosphatase: the hybrid retains both biological activities

We have inserted a disulfide-containing snake neurotoxin intothe N-terminal end of Escherichia coli alkaline phosphatase,between residues +6 and +7 of the mature enzyme. For this purpose,we have designed a cloning and expression vector which allowsinsertion of foreign DNA between the corresponding codons, andvisual selection of the desired recombinant clones upon recoveryof phosphatase activity. The hybrid protein is exported to thebacterial periplasm, the alkaline phosphatase signal peptideis correctly processed, and both domains are functionally conformed.The phosphatase domain displays catalytic activity, and theinserted toxin is able to bind to its biological target, thenicotinic acetylcholine receptor. The hybrid molecule is remarkablystable and resistant to proteolysis. Crude periplasmic extractcontaining the hybrid can be used as a tracer-containing reagentin competitive enzymo-immuno and enzymo-receptor assays. Wepropose to use the system described in this paper for fast preparationof properly folded disulfide-containing enzymatic probes.     

Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins

We have designed and constructed a DNA sequence encoding humantissue plasminogen activator (tPA) with convenient restrictionsites that flank each of the domains of the heavy chain. Toaccomplish this, the first 1095 bases of the gene coding forthe mature protein were synthesized with unique restrictionsites engineered into the interdomainal regions. This syntheticconstruction was then ligated to a cDNA fragment of the tPAgene that encoded the active site, thus generating a full-lengthtPA gene. The gene products produced by Chinese hamster ovary(CHO) cells transfected with either the tPA cassette gene orthe tPA cDNA gene were then compared with the tPA produced byBowes melanoma cells to determine whether or not synthetic interdomainalamino acid changes had an effect on the biochemical characteristicsof the molecule. Specifically, molecular weight, specific activity,enhancement by fibrinogen fragments and kinetic constants wereanalysed. None of the properties examined were significantlydifferent from those of the native melanoma tPA. Therefore,the cassette gene described herein should provide considerableversatility and precision in the construction of tPA mutantsby facilitating the manipulation of the finger, growth factorand kringle domains, and likewise should be useful in assessingthe function of these domains within the tPA molecule. We presentthis cassette gene system as a model for the analysis of proteindomain function applicable to other multi-domain proteins.     

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.     

Nuclear magnetic resonance chemical shift: comparison of estimated secondary structures in peptides by nuclear magnetic resonance and circular dichroism

Traditionally, CD has been used extensively for peptides insecondary structure analysis. In recent years, NMR chemicalshifts and nuclear Overhauser enhancements have been widelyused in conjunction with CD to assess the secondary structuresof peptides and proteins; however, there are many instanceswhere the estimation of secondary structure contents differssignificantly between the two methods. In order to elucidatethe perceived differences between the two methods, secondarystructure estimations by CD and ¹H NMR chemical shifts werecompared for over 50 peptides. The linear peptides investigatedwere largely unstructured, {small tilde}15–50 residuesin size, and lacked stable tertiary conformation. These peptideswere studied in different solvent systems including water, alcohol—water,micelles and urea. A strong correlation exists for secondarystructure assessment by CD and NMR chemical shifts; however,an interesting trend of higher estimation of helical contentsby NMR was observed for peptide fragments from globular proteinsstudied in water. This may be a result of associative propertiesof these peptides in water. Addition-ally, a new method of quantitatingsecondary structure contents based on ¹H NMR chemical shiftsis reported.     

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.     

Mammalian cell transient expression of tissue factor for the production of antigen

We describe a mammalian cell expression system used to rapidlyproduce microgram quantities of a membrane protein used as animraunogen. A fusion protein expression vector was constructedwhich contained the signal sequence and 27 amino acids of theHerpes simplex virus glycoprotein D (gD), followed by a factorVIII (fVIII) thrombin cleavage site and the mature tissue factor(TF) sequence. This fusion protein was transiently expressedand then purified using an antibody to gD. The purified fusionprotein, gDTF, was incubated with thrombin to remove the gD-fVIIImoiety and the resulting rTF served as antigen for the generationof TF-specific antibodies. The antibodies produced were thenused for a comparison of the turnover rates of the constitutivelyand transiently produced fusion protein. In addition, sensitivityto glycosidases indicated that the transiently and constitutivelyproduced recombinant proteins do not contain identical carbohydratestructures.     

Using archaeal histones for precise DNA fragmentation

The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of approximately 60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.     

Design of fully active FGF-1 variants with increased stability

Fibroblast growth factor 1 is a powerful mitogen playing an important role in morphogenesis, angiogenesis and wound healing and is therefore of potential medical interest. Using homologous sequence and structure comparisons, we designed and constructed 16 mutants of FGF-1 with increased thermodynamic stability, as determined by chemical and heat denaturation. For multiple mutants, additive effects on stability were observed, providing mutants up to 7.8 degrees C more stable than the wild-type. None of the introduced mutations affected any FGF-1 biological activities, such as stimulation of DNA synthesis, MAP kinase activation and binding to the FGF receptor on the cell surface. Our study provides a good starting point to improve the stability of FGF-1 in the context of its wide potential therapeutic applications. We showed that a homology approach is an effective method to change the thermodynamic properties of the protein without altering its function.     

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.     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes