Engineering a calcium-dependent conformational change in Calbindin D9k by secondary elements replacement.

EF-hand is a common motif in Ca2+-binding proteins, some of which present a conformational change upon Ca2+-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D9k, a modulator protein with a high affinity for Ca2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D9k. Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca2+ affinity and restricting motif conformation.

Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to lepidopteran toxicity.

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression.

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly.

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named "Spiked Genes" yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.

High-level expression in Escherichia coli, purification and kinetic characterization of Plasmodium falciparum M1-aminopeptidase.

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1mM IPTG at 37°C for 18h. This allowed obtaining 100mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn(2+) excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors.

Combinatorial multicomponent access to natural-products-inspired peptidomimetics: discovery of selective inhibitors of microbial metallo-aminopeptidases.

The development of selective inhibitors of microbial metallo-aminopeptidases is an important goal in the pursuit of antimicrobials for therapeutic applications. Herein, we disclose a combinatorial approach relying on two Ugi reactions for the generation of peptidomimetics inspired by natural metallo-aminopeptidase inhibitors. The library was screened for inhibitory activity against the neutral metallo-aminopeptidase of Escherichia coli (ePepN) and the porcine kidney cortex metallo-aminopeptidase (pAPN), which was used as a model of the M1-aminopeptidases of mammals. Six compounds showed typical dose-response inhibition profiles toward recombinant ePepN, with two of them being very potent and highly selective for ePepN over pAPN. Another compound showed moderate ePepN inhibition but total selectivity for this bacterial enzyme over its mammalian orthologue at concentrations of physiological relevance. This strategy proved to be useful for the identification of lead compounds for further optimization and development.

A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level.

The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca(2+)-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca(2+)-dependent conformational change. The Ca(2+)-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca(2+)-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca(2+) conditions. Because TyrA activity exhibits robust changes in response to Ca(2+)-dependent-EF-hand conformational alterations, TyrA could be a good Ca(2+)-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca(2+)-dependent conformational change.

The ß1 domain of protein G can replace the chorismate mutase domain of the T-protein.

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the ß1-domain of protein G (Gß1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gß1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gß1 domain of Gß1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gß1 substitutions in the N-terminal ß-hairpin eliminated Gß1-TyrA expression, whereas Gß1-TyrA tolerated Gß1 substitutions in the C-terminal ß-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the ß2-strand in forming a Gß1 homo-dimerization interface explains the relevance of the first-ß-hairpin in stabilizing the dimeric TyrA protein.

Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP.

Expression of hypophysiotropic TRH, that controls thyroid axis activity, is increased by cold exposure; this effect is mimicked in rat hypothalamic cells incubated with norepinephrine or cAMP analogs. TRH proximal promoter contains three putative CRE: Site-4 or CRE-1 that overlaps an element recognized by thyroid hormone receptors, CRE-2 with adjacent sequences GC box or CACCC recognized by Sp/Krüppel factors (extended CRE-2), and AP-1 sites flanking a GRE(1/2). To evaluate the role of each element in the cAMP response, these sites were mutated or deleted in rat TRH promoter linked to luciferase gene (TRH-luc) and co-transfected with ß-gal expression vector in various cell lines; C6 cells gave the highest response to forskolin. Basal activity was most affected by mutations or deletion of CRE-2 site, or CACCC (50-75% of wild type-WT). Forskolin-induced 3× stimulation in WT which decreased 25% with CRE-1 or AP-1 deletions, but 50% when CRE-2 or its 5' adjacent GC box was altered. SH-SY5Y cells co-transfected with CREB-expression vector increased dB-cAMP response in the wild type but not in the CRE-2 mutated plasmid; cotransfecting CREB-A (a dominant negative expression vector) strongly diminished basal or cAMP response. Primary cultures of hypothalamic cells transfected with plasmids containing deletions of CRE-1, CRE-2, or extended CRE-2 failed to respond to forskolin when CRE-2 was modified. These results corroborate the CRE-2 site as the main cAMP-response element of rat TRH promoter, not exclusive of transcription factors of hypothalamic cells, and stress the relevance of adjacent Sp-1 sites, important mediators of some metabolic hormones.

Growth rate of a non-fermentative Escherichia coli strain is influenced by NAD+ regeneration.

By complementing a non-fermentative Escherichia coli (ldhA (-) pflB (-)) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD(+) regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.

The effect of amino acid deletions and substitutions in the longest loop of GFP.

BACKGROUND: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. RESULTS: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. CONCLUSION: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

Improvement of an unusual twin-arginine transporter leader peptide by a codon-based randomization approach.

Secretion of Escherichia coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value).

Homology modeling and site-directed mutagenesis of pyroglutamyl peptidase II. Insights into omega-versus aminopeptidase specificity in the M1 family.

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Protein evolution by codon-based random deletions.

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli.

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).

Production of a fully functional, permuted single-chain penicillin G acylase.

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the beta-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the alpha-domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single-chain variants were produced when using a Sec-dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active-site titration experiments showed that the single-chain proteins displayed similar k(cat) values to the ones obtained with the wild-type enzyme. Interestingly, the single-chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection.

Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.

Improving a circularly permuted TEM-1 beta-lactamase by directed evolution.

Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme.