High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcalprotein A, designated domain B, was chemically synthesized.This gene was tandemly repeated to give dimeric and tetramericdomain B genes by the use of two restriction enzymes which gaveblunt ends. The genes were highly expressed in Escherichia colito afford a large amount of dimeric and tetrameric domain Bproteins. The single domain B protein was efficiently producedas a fusion protein with a salmon growth hormone fragment. Thefusion protein was converted to monomeric domain B by cyanogenbromide cleavage. The CD spectra of the monomeric, dimeric andtetrameric domain B proteins were essentially the same as thatof native form protein A, showing that their secondary structureswere very similar. The dimeric and tetrameric domain B proteinsformed precipitates with IgG as protein A. This system permitsthe efficient production of mutated single and multiple IgG-bindingdomains which can be used to study structural changes and proteinA–immunoglobulin interactions.     

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.     

A new family of sugar-inducible expression vectors for Escherichia coli

A set of 11 expression vectors was constructed, each of themharbouring a cloning cassette under the control of the promoter.Some of these vectors enable expression of foreign proteinsin the cytoplasm, while others include a synthetic sequencecoding for a very efficient secretion signal sequence. Otherfeatures are an fl origin of replication (in plus or minus orientation)and a promoter^up mutation that enhances the already very highlevel of expression from these vectors. With such a versatilevector family, cloning, sequencing and sitedirected mutagenesiscan be performed on the same vector, and the level of expressioncan be defined according to the specific constraints of a givenprotein.     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growthmedium is one of the major drawbacks in its use in genetic engineering.A synthetic gene, homologous to the one coding for the kil peptideof pColE1, was made and cloned under the control of the lacpromoter, in order to obtain the inducible secretion of homologousor heterologous proteins by E.coli. The efficiency of this syntheticgene to promote secretion was assayed by analysing the productionand secretion of two proteins, the R-TEM1 ß-lactamase,and the -amylase from Bacillus licheniformis. This latter proteinwas expressed in E.coli from its gene either on the same plasmidas the kil gene or on a different plasmid. The primary effectof the induction of the kil gene is the overproduction of thesecreted proteins. When expressed at a high level, the kil genepromotes the overproduction of all periplasmic proteins andthe total secretion in the culture medium of both the ß-lactamaseor the -amylase. This secretion is semi-selective for most periplasmkproteins are not secreted. The kil peptide induces the secretionof homologous or heterologous proteins in two steps, first actingon the cytoplasmic membrane, then permeabilizing the outer membrane.This system, which is now being assayed at the fermentor scale,is the first example of using a synthetic gene to engineer anew property into a bacterial strain.     

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein

A high-expression plasmid of the canine milk lysozyme, whichbelongs to the family of calcium-binding lysozymes, was constructedin order to study its physico-chemical properties. Because thecDNA sequence of the protein has not yet been determined, a400 base-pair gene encoding canine milk lysozyme was first designedon the basis of the known amino acid sequence. The gene wasconstructed by an enzymatic assembly of 21 chemically synthesizedoligonucleotides and inserted into an Escherichia coli expressionvector by stepwise ligation. The expression plasmid thus constructedwas transformed into BL21(DE3)/pLysS cells. The gene productaccumulated as inclusion bodies in an insoluble fraction. Recombinantcanine milk lysozyme was obtained by purification and refoldingof the product and showed the same characteristics in termsof bacteriolytic activity and far- and near-UV circular dichroismspectra as the authentic protein. The NMR spectra of refoldedlysozyme were also characteristic of a native globular protein.It was concluded that recombinant canine milk lysozyme was foldedinto the correct native structure. Moreover, the thermal unfoldingprofiles of the refolded recombinant lysozyme showed a stableequilibrium intermediate, indicating that the molten globulestate of this protein was extraordinarily stable. This expressionsystem of canine milk lysozyme will enable biophysical and structuralstudies of this protein to be extended.     

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin

A synthetic gene based on the published amino acid sequencefor Clostridium pasteurianum rubredoxin was constructed, clonedin Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promotersystem in E.coli HMS273. UV/visible spectroseopy and metal analysesindicated that the as–isolated synthetic gene productis a mixture of holo–(i.e. iron–containing) rubredoxinand zinc–substituted rubredoxin, with the latter amountingto {small tilde} 70% of the total rubredoxin. Hie UV/visibleabsorption and resonance Raman spectra of the cloned holorubredoxinare characteristic of the native rubredoxin–type ironsite. N–terminal amino acid sequencing suggests that thegene product consists of at least three polypeptide specieswith the initial sequences (approximate relative abundances):Met–Met–Lys–... (63%), blocked (30%) and Met–Lys–...(7%). The blocked portion presumably consists of a mixture ofnMet–Met–Lys–... and nMet-Lys–..., wherenMet represents an amino–blocked methionine residue.     

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.     

Prion protein gene expression in cultured cells

A single copy gene encodes both the scrapie (PrP^Sc) and cellular(PrP^C) isoforms of the prion protein (PrP). Cultured cell lineswere found to express the endogenous PrP mRNA at levels comparableto those observed in the brains of adult rodents; however, thesecells were invariably found to express greatly reduced levelsof PrP. In all the cell lines examined, PrP was undetectableby Western immunoblot analysis. These cells were also poor recipientsfor expression constructs linking the hamster PrP gene openreading frame to several strong eukaryotic promoters; stableclones derived by transfection of these expression vectors failedto show elevated expression of PrP. When extremely high levelsof PrP mRNA were produced using either an insect baculovirusor a mammalian SV40 based vector, significant quantities ofPrP were produced, although in both cases the proteins wereapparently processed differently from the PrP^C observed in brains.In an expression system using an SV40 late promoter vector inmonkey COS-7 cells, a significant fraction of PrP was transportedto the cell surface where PrP^C is found in vivo. PrP synthesizedby the baculovirus vector failed to induce scrapie in hamstersand did not possess the characteristics of the PrP^Sc isoformassociated with infectivity. The SV40 late promoter vector systemmay permit experiments designed to elucidate the role of PrP^Scduring scrapie infection as well as the function of PrP^C innormal metabolism.     

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.     

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.     

Greek key jellyroll protein motif design: expression and characterization of a first-generation molecule

A protein designed de novo to fold into the Greek key jellyrollstructural motif has been studied. Theoretical analyses haveindicated that the designed sequence should adopt the ß-strandarrangement of the Greek key jellyroll rather than any otherarrangement. A synthetic gene was constructed and the proteinexpressed in Escherichia coli. Circular dichroism spectroscopyis consistent with the protein folding into the designed conformationand also suggests the presence of tertiary structure. Fluorescencespectroscopy showed the single tryptophan to be partially buried,while denaturation studies showed changes in fluorescence toprecede alterations in secondary structure.     

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.     

The construction and cloning of synthetic genes coding for artificial proteins and expression studies to obtain fusion proteins

Synthetic genes coding for artificial proteins with predeflnedand nutritionally valuable amino acid compositions have beenconstructed and cloned In bacterial plasmid vector pKK233-2.The genes were constructed from three easily interchangeable‘cassettes’ encoding either essential, non-essentialor branched-chain amino acid residues. A potential hairpin loopstructure in the mRNA around the region of the ribosome bindingsite was probably the reason for blockage of translation fromthis vector. Two selected genes, AHB (containing one copy ofeach cassette) and A (consisting of six copies concatemerizedA₆cassette) were cloned into pUR300, a (ß-Gal fusionvector and expressed as fusion proteins (ß-Gal-AHBand (ß-Gal-A₆.     

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     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-gal–proAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor X_a. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-gal–proAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor X_a digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorX_a, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDS–polyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled.     

Overexpression and structure-function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineera bifunctional lymphokine which was overproduced in Escherichiacoli. Following denaturation of the inclusion bodies in 6 Mguanidine and refolding and reoxidation in the presence of aredox system, the fusion protein (rIL-2/IL-6) was purified tohomogeneity and shown to react with both monospecific anti-IL-2and anti-IL-6 antisera. A collagen-like spacer was introducedbetween the two cytokine moieties to generate IL-2 and IL-6molecules upon collagenase digestion. After cleavage, the twosubunits, purified in a single-step procedure, were found tobe correctly reoxidized and functionally as active as theirnative counterparts. Circular dichroism studies of rIL-2/IL-6revealed that both cytokine subunits refolded independentlyand exhibited the a-helical structures characteristic of thecorresponding wild-type lymphokines. The chimera displayed fullIL-2 activity in the CTLL-2 cell proliferation assay. It alsoretained the IL-6 property to enhance lgM synthesis in SKW6.4cells, induce the proliferation of B-cell hybridomas and stimulatethe production of fibrinogen in hepatocytes. Because IL-2 amplifiesthe cellular immune response and IL-6 up-regulates the humoralresponse, this bifunctional lyinphokine represents a potentiallyuseful therapeutic adduct and may serve as an immunomodulatorto enhance the host's response to vaccination.     

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})     

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.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis

A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-aminopenicillanic acid (6-APA) and activated D-phenylglycine derivatives. We directed changes in amino acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: alpha R160, alpha F161 and beta F24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method. A detailed kinetic analysis of the three most promising mutants, T23, TM33 and TM38, is presented. These mutants allowed the accumulation of ampicillin at 4-5 times higher concentrations than the wild-type enzyme, using D-phenylglycine methyl ester as the acyl donor. At the same time, the loss of activated acyl donor due to the competitive hydrolytic side reactions could be reduced to <20% with the mutant enzymes compared >80% wild-type PAS2. Although catalytic activity dropped by a factor of 5-10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of beta-lactam antibiotics.