Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins

The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.     

Problem of proteolytic enzyme evolution

Own and literature data on putative evolution of proteolytic enzymes have been reviewed. Modern principles of peptidase classification based on evaluation of homology of more than 66 thousand gene sequences and similarity of general structural organization of almost 2.5 thousands of known enzymes are considered. The review highlights not only evolutionary interrelationships inside related peptidase families, their possible evolutionary background, but also evolutionary determined differences in certain proteolytic pathway in organisms belonging to different taxons.     

Directed enzyme evolution

Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.     

核糖体展示及体外分子选择与进化

核糖体展示是20世纪90年代中期发展起来的一种简便而有效的体外分子选择与进化技术。它也是第一种完全在体外进行蛋白质或多肽分子选择与进化的方法。本主要概述了体外核糖体展示技术的建立基础、基本原理和技术特点等,并跟踪了目前该领域的最新研究进展和发展前景。     

Activity-based selection of a proteolytic species using ribosome display

We have examined the potential of displaying a protease species in vitro using ribosome display and demonstrate specific capture on the basis of its catalytic activity. Using a model bacterial cysteine protease, sortase A (SrtA), we show that this enzyme can be functionally expressed in vitro. By overlap PCR we constructed ribosome display templates with the SrtA open reading frame fused to a C terminal glycine-serine rich flexible linker and a tether derived from eGFP. Using the broad range cysteine protease irreversible inhibitor E-64 linked to acrylic beads, we show that we can isolate SrtA ribosome display ternary complexes, and recover their encoding mRNA by RT-PCR. This recovery was lost when applied to a SrtA catalytically inactive mutant, or could be alleviated by competition with free inhibitor. This sensitive technique could be further developed to allow the screening of proteases against putative inhibitors and/or the identification of novel proteolytic species.     

基于易错PCR的黄曲霉毒素解毒酶体外分子定向进化

运用定向进化-易错PCR方法,提高黄曲霉毒素解毒酶的活力及稳定性,并结合辣根过氧化物酶 (HRP)-隐性亮绿 (RBG) 快速高通量筛选系统,构建了库容约为104的突变体库。经过两轮易错PCR,最终分别获得了耐高温70 ℃突变酶A1773、pH 4.0稳定性的突变酶A1476,pH 4.0和pH 7.5均表现稳定性的突变酶A2863,其酶活力比野生酶分别提高了6.5倍、21倍和12.6倍。经序列分析表明,发现突变酶A1773发生了Glu127Lys和Gln613Arg突变;突变酶A2863发生了Gly73     

Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: (1) those that disrupt packing of hydrophobic side chains in the protein interior; (2) those that destabilize secondary structure; and (3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2–5 kcal/mol. © 1995 Wiley-Liss, Inc.     

Directed evolution of novel protein functions

Directed evolution has been successfully used to engineer proteins for basic and applied biological research. However, engineering of novel protein functions by directed evolution remains an overwhelming challenge. This challenge may come from the fact that multiple simultaneously or synergistic mutations are required for the creation of a novel protein function. Here we review the key developments in engineering of novel protein functions by using either directed evolution or a combined directed evolution and rational or computational design approach. Specific attention will be paid to a molecular evolution model for generation of novel proteins. The engineered novel proteins should not only broaden the range of applications of proteins but also provide new insights into protein structure-function relationship and protein evolution.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

R-2-氯丙酸脱卤酶的定向进化

通过易错PCR手段将R-2-氯丙酸脱卤酶定向进化,并使用基于Cl-浓度显色反应的高通量筛选得到有效突变子库,发现突变子DehDIV-G2和DehDIV-E7的酶比活力分别提高25.2%和38.7%。通过SYBYL对酶与底物进行分子对接显示,DehDIV-G2的活化能下降0.961 4 kJ/mol,DehDIV-E7的活化能下降2.549 8 kJ/mol。由于酶和底物R-2-氯丙酸的活化能下降,亲和能力提高,从而提高酶的比活力。     

Origins of the high stability of an in vitro-selected cold-shock protein

In previous work, we had identified stabilized forms of the cold-shock protein Bs-CspB from Bacillus subtilis in a combinatorial library by an in vitro selection procedure. In this library, the sequence positions 2, 3, 46, 64, 66, and 67 had been randomized, because Bs-CspB differs from the naturally thermostable homolog Bc-Csp from Bacillus caldolyticus, among others, at these six positions. For the most stable selected variant, the midpoint of thermal unfolding (tM) increased by 28.2 deg. C and the Gibbs free energy of unfolding (deltaG(D)) by 19 kJ/mol. Here, we analyzed by site-directed mutagenesis how the selected residues contribute individually to this strong stabilization. Val3 and Val66, which replace Glu3 and Glu66 of wild-type Bs-CspB, each contribute about 7 kJ/mol to stability, the Thr64Arg substitution contributes 4.5 kJ/mol, and 3.2 kJ/mol originate from the Ala46Leu replacement. Gly67 at the carboxy terminus is unimportant for stability, the Arg selected at position 2 is overall slightly destabilizing but improves the coulombic interactions. The best variant differs from Bc-Csp at all six positions; nevertheless, natural and in vitro selection followed similar principles. In both cases, negatively charged residues at the adjacent positions 3 and 66 are avoided, and a positively charged residue is introduced into this area of the protein surface. Its exact location is unimportant. It can be at position 3, as in the thermophilic Bc-Csp, or at positions 2 or 64, as in the most stable selected variant. These positively charged residues contribute to stability not by engaging in pairwise coulombic interactions with a specific carboxyl group, but by generally improving the charge distribution in this particular region of the protein surface. These coulombic effects contribute significantly to the thermostability of the cold-shock proteins. They are only weakly interdependent and best explained by the presence of a flexible ion network at the protein surface. Our results emphasize that surface positions are very good candidates for optimizing protein stability.     

定向进化细胞色素P450BM-3的研究

以来自于巨大芽孢杆菌的细胞色素P450BM-3为研究对象,采用随机突变和饱和定点突变定向进化技术对P450BM-3进行改造,通过突变体催化靛蓝显色的特性采用活性琼脂平板分析和96微孔板相结合的高通量筛选成功获得了几个具有更高催化性能的突变体。     

蛋白质定向进化的研究进展

在工业生物催化过程和生物细胞工厂构建方面,蛋白质定向进化被广泛地应用于酶的分子改造.蛋白质定向进化不仅可以针对某一目的蛋白进行改造,还可以改善代谢途径、优化代谢网络、获得期望表型细胞.为了获得更高效的突变效率,快捷、高通量的筛选方法,提高蛋白质定向进化的效果,研究者不断开发蛋白质体内、体外进化方法,取得了新的进展和应用.本文介绍了最近发展的蛋白质定向进化技术的原理、方法及特点,总结了突变文库的筛选方法和蛋白质定向进化的最新应用,最后讨论了蛋白质定向进化存在的挑战和未来发展方向.     

Fertility selection,genetic selection and evolution

The relationship between fertility selection as measured by the correlation in progeny number between parents and offspring, and selection at individual loci is investigated in humans. Estimates for the magnitude of fertility selection (0.1) and the rate of gene substitution (0.5 gene substitutions per generation per genome) are used in various mathematical models for selection. It is found that the observed magnitude of fertility selection cannot be explained by non‐epistatic directional selection at individual loci. A symmetric quantitative directional selection model is consistent with the observed data. But it is possible that fertility selection does not have a genetic basis.     

Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase

The Escherichia coli aspartate (AATase) and tyrosine (TATase) aminotransferases share 43% sequence identity and 72% similarity, but AATase has only 0.08% and 0.01% of the TATase activities (k(cat)/K(m)) for tyrosine and phenylalanine, respectively. Approximately 5% of TATase activity was introduced into the AATase framework earlier both by rational design (six mutations, termed HEX) and by directed evolution (9-17 mutations). The enzymes realized from the latter procedure complement tyrosine auxotrophy in TATase deficient E. coli. HEX complements even more poorly than does wild-type AATase, even though the (k(cat)/K(m)) value for tyrosine exhibited by HEX is similar to those of the enzymes found from directed evolution. HEX, however, is characterized by very low values of K(m) and K(D) for dicarboxylic ligands, and by a particularly slow release for oxaloacetate, the product of the reaction with aspartate and a TCA cycle intermediate. These observations suggest that HEX exists largely as an enzyme-product complex in vivo. HEX was therefore subjected to a single round of directed evolution with selection for complementation of tyrosine auxotrophy. A variant with a single amino acid substitution, A293D, exhibited substantially improved TATase function in vivo. The A293D mutation alleviates the tight binding to dicarboxylic ligands as K(m)s for aspartate and alpha-ketoglutarate are >20-fold higher in the HEX + A293D construct compared to HEX. This mutation also increased k(cat)/K(m)(Tyr) threefold. A second mutation, I73V, elicited smaller but similar effects. Both residues are in close proximity to Arg292 and the mutations may function to modulate the arginine switch mechanism responsible for dual substrate recognition in TATases and HEX.     

甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖

甲壳素酶具有广泛的工业应用前景,如可将虾壳、蟹壳和其他甲壳废物降解成以几丁寡糖为主的高附加值产品,但野生型甲壳素酶催化效率低,大大限制了几丁寡糖的生产。笔者在前期研究中表达了一个具有较高效催化效率的甲壳素酶Chisb,并对其酶学性质进行了初步研究。为进一步提高甲壳素酶Chisb的催化效率,以R13NprB-C-SP-H为亲本,采用易错PCR(Error-pronePCR)技术构建随机突变体文库,对甲壳素酶Chisb进行定向进化。经过96孔板初筛和摇瓶复筛,获得了两个催化效率进一步提高的突变体C43D和E336R。对突变体的酶学性质进行分析, C43D和E336R的最适催化温度为55℃, C43D的最适pH为5.0,E336R的最适pH为9.0;其催化效率相比对照分别提高了1.35倍和1.57倍;而E336R和C43D催化产几丁寡糖的含量分别为2.53 g/L和2.06 g/L,相比对照(0.89 g/L)分别提高了2.84倍和2.31倍;底物转化率分别为84.3%和68.7%,相比对照(29.7%)分别提高了54.6%和39%。研究表明,通过易错PCR引入随机突变的方法能够有效提高甲壳素酶Chisb的催化效率。上述研究获得的催化效率提高的正向突变体及其酶学性质分析对生物转化合成几丁寡糖具有重要研究意义和应用价值。     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.