Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

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.     

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the productof the Escherichia coli aroA gene, has been overproduced inE.coli BL21(DE3) under the control of the T7 gene 10 promoterand ribosome binding site, to a level of {small tilde}50% oftotal cell protein. EPSP synthase is the primary target of thepost-emergence herbicide, glyphosate, commonly known as Roundup^TM.A simple two step purification is described, which results in99% pure homogeneous protein (as determined by PAGE). The integrityof the protein has been compared with previously characterizedprotein from .E.coli AB2829(pKD501) by determination of itskinetic parameters, N-terminal protein and DNA sequences, aminoacid analysis and ¹³C-NMR spectroscopy. This new overproducingstrain readily provides the gram quantities of highly pure proteinrequired for NMR studies of the active site and the developmentof novel time-resolved solid-state NMR techniques currentlyunderway in this laboratory.     

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.     

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

Bovine pancreatic ribonuclease A (RNase A) has been the objectof much landmark work in biological chemistry. Yet the applicationof the techniques of protein engineering to RNase A has beenlimited by problems inherent in the isolation and heterologousexpression of its gene. A cDNA library was prepared from cowpancreas, and from this library the cDNA that codes for RNaseA was isolated. This cDNA was inserted into expression plasmidsthat then directed the production of RNase A in Saccharomycescerevisiae (fused to a modified -factor leader sequence) orEscherichia coli (fused to the pelB signal sequence). RNaseA secreted into the medium by S.cerevisiae was an active buthighly glycosylated enzyme that was recoverable at 1 mg/l ofculture. RNase A produced by E.coli was in an insoluble fractionof the cell lysate. Oxidation of the reduced and denatured proteinproduced active enzyme which was isolated at 50 mg/l of culture.The bacterial expression system is ideal for the large-scaleproduction of mutants of RNase A. This system was used to substitutealanine, asparagine or histidine for Gln11, a conserved residuethat donates a hydrogen bond to the reactive phosphoryl groupof bound substrate. Analysis of the binding and turnover ofnatural and synthetic substrates by the wild-type and mutantenzymes shows that the primary role of Gln11 is to prevent thenon-productive binding of substrate.     

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.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Relationship between thermal stability and 3-D structure in a homology model of 3-isopropylmalate dehydrogenase from Escherichia coli

To reveal the structural basis of the increased thermal stabilityof 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus,an extreme thermophile, the homology-based structural modelof one mesophilic (Escherichia coli) counterpart, was constructed.Both IPMDHs are homodimeric proteins. We built a model of onesubunit using the 3-D structures of the Th.thermophilus IPMDHand the homologous E.coli isocitrate dehydrogenase. Energy minimizationand molecular dynamics simulated annealing were performed onthe dimer, including a surrounding solvation shell. No seriouserrors were detected in the refined model using the 3-D profilemethod. The resulting structure was scrutinized and comparedwith the structure of the Th.thermophilus IPMDH. Significantdifferences were found in the non-specific interactions includingthe hydrophobic effect. The model predicts a higher number ofion pairs in the Th.thermophilus than hi the E.coli enzyme.An increase was observed in the stabilities of -helical regionshi the thermophilic protein. The preliminary X-ray coordinatesof the E.coli IPMDH were received after the completion of thiswork, allowing an assessment of the model in terms of the X-raystructure. The comparison proved that most of the structuralfeatures underlying the stability differences between the twoenzymes were predicted correctly.     

Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli

Recombinant porcine myoglobin has been produced in Escherichiacoli using the cII fusion expression system of Nagai and Th?gersen[Nature, 309, 810–812 (1984)]. After processing and reconstitutionwith haem, the protein is gel-electrophoretically and spectrophotometricallyindistinguishable from native pig myoglobin. Large crystalsof both native and recombinant porcine myoglobin were grownfrom 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate.The crystals belong to space group C2 (a = 156.9 ?, b = 42.0?, c = 92.2 ?, ß = 127.9?) and diffract to a nominal2.5 ? resolution. We plan to explore apomyoglobin as a bindingsurface in studies combining site-directed mutagenesis and X-rayanalysis. These experiments will be extended by studying thebinding of haem analogues to the mutant apoproteins.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

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.     

Synthesis and expression in Escherichia coli of DNA encoding the murine {lambda}1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen

A 658 bp DNA sequence corresponding to the murine ₁ chain ofa monoclonal antibody, Se 155-4, specific for the Salmonellaserotype B O-antigen, was designed using Escherichia coli preferredcodons and chemically synthesized by ligation of synthetic fragmentsinto a linearized plasmid followed by transformation into E.coli.A synthetic signal peptide (ompA) was fused to express the Lchain as a free polypeptide into the periplasm of E.coli cells.After isolation and purification, heterologous recombinationof the E.coli L chain with mouse H chain gave an active antigen-bindingprotein. The activity was 15–20% when compared to proteincreated by an equivalent association of isolated natural mouseL and H chains as measured by a direct EIA assay. In inhibitionexperiments with the polysaccharide antigen, the two proteinsshowed identical titration curves and 50% inhibition points,indicating comparable K_A values.     

Design of four-helix bundle protein as a candidate for HIV vaccine

To be efficient, a synthetic vaccine should contain differentT and B cell epitopes of human immunodeficiency virus (HIV)antigens, and the B epitope regions in the vaccine and in theHIV should be conformationally similar. We have suggested previouslythe construction of vaccines in the form of a protein with apredetermined tertiary structure, namely a four--helix bundle.Antigenic determinants of cellular and humoral immunity areblocks for the vaccine design. From experimentally studied HIV-1T and B cell epitopes, we constructed a sequence of a four-helixprotein TBI (T and B cell epitopes containing immunogen). Thegene of the protein was synthesized and the protein was producedin C600 Escherichia coli cells under recA promoter from Proteusmirabelis. CD spectroscopy of the protein demonstrated that30% of amino acid residues adopt an -helical conformation. Miceimmunized with TBI have shown both humoral and cellular immuneresponses to HIV-1. The obtained data show that the design ofTBI was successful. The synthesized gene structure makes possiblefurther reconstruction and improvement of the protein vaccinestructure.     

A combinatorial library of an {alpha}-helical bacterial receptor domain

The construction and characterization of a combinatorial libraryof a solvent-exposed surface of an -helical domain derived froma bacterial receptor is described. Using a novel solid-phaseapproach, the library was assembled in a directed and successivemanner utilizing single-stranded oligonucleotides containingmultiple random substitutions for the variegated segments ofthe gene fragment The simultaneous substitution of 13 residuesto all 20 possible amino acids was carried out in a region spanning81 nucleotides. The randomization was made in codons for aminoacids that were modelled to be solvent accessible at a surfacemade up from two of the three a-helices of a monovalent Fc-bindingdomain of staphylococcal protein A. After cloning of the PCR-amplifiedlibrary into a phagemid vector adapted for phage display ofthe mutants, DNA sequencing analysis suggested a random distributionof codons in the mutagenized positions. Four members of thelibrary with multiple substitutions were produced in Escherichiacoli as fusions to an albumin-binding affinity tag derived fromstreptococcal protein G. The fusion proteins were purified byhuman serum albumin affinity chromatography and subsequentlycharacterized by SDSelectrophoresis, CD spectroscopy and biosensoranalysis. The analyses showed that the mutant protein A derivativescould all be secreted as soluble full-length proteins. Furthermore,the CD analysis showed that all mutants, except one with a prolineintroduced into helix 2, have secondary structures in closeagreement with the wild-type domain. These results proved thatmembers of this -helical receptor library with multiple substitutionsin the solvent-exposed surface remain stable and soluble inE.coli. The possibility of using this library for a phenotypicselection strategy to obtain artificial antibodies with novelfunctions is discussed.     

Overproduction and purification of the regulatory subunit of Escherichia coli aspartate transcarbamoylase

An Escherichia coli strain/plasmid system has been developedfor the overexpression of the regulatory subunit of E.coli aspartatetranscarbamoylase (ATCase). Production of large quantities ofregulatory subunit, by the method described here, should facilitatefuture experiments, such as X-ray crystallography, NMR and hybridizationexperiments, aimed at understanding the heterotropic mechanismthat regulates the activity of ATCase. The plasmid used forthe over-expression carries the gene for the regulatory subunit,pyrI, downstream from the strong pyrB promoter. The host strain,EK1104 [Nowlan, S.F. and Kantrowitz, E.R. (1985) J. Biol. Chem.,260, 14712–14716] carries a deletion in the pyrBI regionof the chromosome, as well as a leaky pyrF allele. When thisstrain/plasmid system is grown under limiting pyrimidine levels,large quantities of the regulatory subunit of ATCase are producedwithout any trace of catalytic subunit or holo-enzyme. A procedurefor the purification of the regulatory subunit from cell extractshas also been developed yielding {small tilde}50 mg of purifiedregulatory subunit per liter of initial culture. The regulatorysubunit produced in this fashion is fully competent in reassociationexperiments with the native catalytic subunit. Furthermore,the reassociated holoenzyme exhibits kinetic properties identicalto those of the wild type enzyme. In addition, we report theconstruction of a pUC119 based plasmid which carries a uniqueNdeI site at the fMet of the pyrB gene of ATCase. This plasmid,which was used in the construction of the system for the overexpressionof the regulatory subunit of ATCase, has been shown to be ofgeneral use for the expression of foreign proteins in E.coli.     

Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

Overproduction, preparation of monoclonal antibodies and purification of E.coli asparagine synthetase A

In order to explore the structure-function relationship of theEscherichia coli asparagine synthetase A it was necessary todevise a system for overexpression of the gene and purificationof the gene product. The E.coli asparagine synthetase A structuralgene was fused to the 3' end of the human carbonic anhydraseII structural gene and overexpressed in E.coli. The gene product,a 66 kDa fusion protein, which exhibited asparagine synthetaseactivity, was purified in a single step by affinity chromatographyand used as the antigen for the production of monoclonal antibodies.The monoclonal antibodies were screened by ELISA. Colonies werechosen which were positive for purified fusion protein and negativefor purified human carbonic anhydrase II. The E.coli asparaginesynthetase A gene was then overexpressed and the gene productwas used without purification for the final screen. The antibodiesselected were used for immunoaffinity chromatography to purifythe recombinant overexpressed E.coli asparagine synthetase A.Thus, a procedure is now available so that asparagine synthetaseA can be purified to homogeneity in a single step.     

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.     

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.