T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli.

Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction. From the earliest days, basal expression prevented establishment of inducible strains for producing proteins that are stressful to the host. Newly developed pAL vectors now reduce basal expression to levels where coding sequences for even the most stressful proteins can be maintained and induced. Asymmetric ligation allows simple and efficient cloning of individual coding sequences or simultaneous cloning of two or three coding sequences for co-expression from a single pAL vector. © 2018 by John Wiley & Sons, Inc.

Recombinant transfer in the basic genome of Escherichia coli.

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40-115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4-1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.

Stable expression clones and auto-induction for protein production in E. coli.

Inducible production of proteins from cloned genes in E. coli is widely used, economical, and effective. However, common practices can result in unintended induction, inadvertently generating cultures that give poor or variable yields in protein production. Recipes are provided for (1) defined culture media in which expression strains grow to saturation without induction, thereby ensuring stable frozen stocks and seed cultures with high fractions of fully inducible cells, and (2) defined or complex media that maintain the same high fraction of inducible cells until auto-induction in late log phase to produce fully induced high-density cultures at saturation. Simply inoculating a suitable auto-inducing medium from such a seed culture and growing to saturation generally produces much higher levels of target protein per volume of culture than monitoring culture growth and adding IPTG or other inducer at the appropriate cell density. Many strains may be conveniently screened in parallel, and burdensome inoculation with fresh colonies, sometimes employed in hopes of assuring high yields, is entirely unnecessary. These media were developed for the T7 expression system using pET vectors in BL21(DE3) but are suitable or adaptable for other inducible expression systems in E. coli and for labeling proteins with selenomethionine for X-ray crystallography or with stable isotopes for NMR.

Tracing ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606 and BL21(DE3).

Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.

Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes.

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations ( approximately 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrück and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although approximately 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had approximately 4039 coding sequences occupying approximately 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was identified. Complex deletions (dense clusters of small deletions and substitutions) apparently removed nonessential genes from approximately 30 sites in the basic genomes.

Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the lambda attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have >99% sequence identity over approximately 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.

Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase.

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.

Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target.

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.

New York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center for the protein structure initiative.

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.

Protein production by auto-induction in high density shaking cultures.

Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).

Structure and mechanism of ADP-ribose-1''-monophosphatase (Appr-1''-pase), a ubiquitous cellular processing enzyme.

Appr-1''-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1''monophosphate (Appr-1''-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 A and 2.05 A, respectively. Analysis of the three-dimensional structure of this protein, selected as a target in a structural genomics project, reveals its putative function and provides clues to the catalytic mechanism. The structure of the 284-amino acid protein shows a two-domain architecture consisting of a three-layer alphabetaalpha sandwich N-terminal domain connected to a small C-terminal helical domain. The structure of Appr-1''-pase in complex with the product, ADP-ribose, reveals an active-site water molecule poised for nucleophilic attack on the terminal phosphate group. Loop-region residues Asn 80, Asp 90, and His 145 may form a catalytic triad.

Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection.

T7 RNA polymerase selectively transcribes T7 genes during infection but is also involved in DNA replication, maturation and packaging. T7 lysozyme is an amidase that cuts a bond in the peptidoglycan layer of the cell wall, but it also binds T7 RNA polymerase and inhibits transcription, and it stimulates replication and packaging of T7 DNA. To better understand the roles of these two proteins during T7 infection, mutants of each were constructed or selected and their biochemical and physiological behavior analyzed. The amidase activity of lysozyme is needed for abrupt lysis and release of phage particles but appears to have no role in replication and packaging. The interaction between polymerase and lysozyme stimulates both replication and packaging. Polymerase mutants that gain the ability to grow normally in the absence of an interaction with lysozyme still fail to shut down late transcription and, remarkably, have become hypersensitive to inhibition when lysozyme is able to bind. These lysozyme-hypersensitive polymerases behave without lysozyme similarly to wild-type polymerase with lysozyme: both remain longer at the promoter before establishing a lysozyme-resistant elongation complex and both increase the length of pausing when elongation complexes encounter an eight-base recognition sequence involved in DNA packaging. Replication origins contain T7 promoters, but the role of T7 RNA polymerase in initiating replication is not understood well enough to more than speculate how the lysozyme-polymerase interaction stimulates replication. Maturation and packaging is apparently initiated through interaction between prohead-terminase complexes and transcription elongation complexes paused at the sequence TATCTGT(T/A), well conserved at the right-end of the concatemer junction of T7-like phages. A model that is consistent with the structure of an elongation complex and a large body of mutational and biochemical data is proposed to explain sequence-specific pausing and potential termination at the consensus recognition sequence (C/T)ATCTGT(T/A).

Crystal structure of a putative CN hydrolase from yeast.

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.