Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.     

Ligation site in proteins recognized in silico

Recognition of a ligation site in a protein molecule is important for identifying its biological activity. The model for in silico recognition of ligation sites in proteins is presented. The idealized hydrophobic core stabilizing protein structure is represented by a three-dimensional Gaussian function. The experimentally observed distribution of hydrophobicity compared with the theoretical distribution reveals differences. The area of high differences indicates the ligation site. AVAILABILITY: http://bioinformatics.cm-uj.krakow.pl/activesite.     

Ligation of expressed protein α-hydrazides via genetic incorporation of an α-hydroxy acid

Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein α-hydrazides as ligation intermediates that are more chemically stable than protein α-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase(PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNACUAPyl system is compatible with both prokaryotic and eukaryotic cells,the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.     

类泛素蛋白--SUMO

SUMO(small ubiquitin-related modifier)是泛素(ubiquitin)类蛋白家族的重要成员之一。尽管SUMO的生化反应途径与泛素相似,但不像泛素那样诱导底物蛋白降解。SUMO化能够使蛋白质更加稳定,进而调节许多关键的细胞活动。现从分类、结构、生化途径和生物学功能等方面介绍SUMO及SUMO化过程。     

Chemical protein synthesis

Since the mid-1990s, chemical synthesis has emerged as a powerful technique for the study of structure/function relationships in proteins. During the review period, the applicability of chemical protein synthesis techniques has been significantly broadened by increases in the size of synthetically accessible proteins through two new techniques: solid-phase protein synthesis and expressed protein ligation. Also in the period under review, synthetic access to novel classes of proteins has been established, including metalloproteins with tuned properties and integral membrane proteins.     

Side-chain assisted ligation in protein synthesis

Chemical ligation methods for the assembly of functional proteins continue to advance our basic understanding of protein structure and function. In this work, we report on our progress towards the full synthesis of HIV-1 Tat utilizing our newly developed ligation method; side-chain assisted ligation. The HIV-1 Tat was assembled from three fragments wherein the two thioester peptides were synthesized efficiently using the side-chain anchoring strategy following Fmoc-SPPS. The side-chain assisted ligation step was efficient and provided the ligation product in good yield. Following this step, native chemical ligation was used to fully assemble the HIV-1 Tat protein. Although the removal of the auxiliary in small peptides was straightforward, in the case of HIV-1 Tat this step was inefficient thus hampering the completion of the synthesis.     

Total synthesis of artificial zinc-finger proteins: Problems and perspectives

The total synthesis of a peptide segment corresponding to the DNA-binding segment of Sp1 (positions 532-623) using a native chemical ligation approach is described. The folding of the synthetic segment in the presence of Zn(II) gave a zinc-coordinated protein. The dissociation constant (K(D)) for the DNA binding of the resulting protein, determined by a gel mobility shift assay, was 130 nM, almost nine times higher than that of the genetically prepared protein. However, methylation interference assay showed an identical sequence specificity of both proteins in DNA recognition. The chemical ligation method to connect the respective zinc-finger units was also accomplished. Successive ligation between a cysteine-containing peptide segment and a chloroacetylated peptide segment gave an artificial three-finger protein, which corresponds to the above DNA-binding segment of Sp1. However, this protein failed to bind DNA, even at 1.25 mM. Assessment of their folding structure based on the absorption spectra of their Co(II) complexes showed that the linker design to connect the respective finger units is critical for the proper folding of the proteins as well as the occurrence of the DNA-binding function.     

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys. Similarly, c-Crk-II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of (15)N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems.     

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides for in vitro applications

Understanding the structure and function of protein complexes and multi‐domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon S ortase‐mediated and Na tive chemical ligation using synthetic Pe ptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps – sortase‐mediated and native chemical ligation – together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

GYF结构域蛋白特性及其生物学功能研究进展

甘氨酸-络氨酸-苯丙氨酸(glycine-tyrosine-phenylalanine,GYF)结构域是一种小型的、能识别富脯氨酸序列(proline rich sequences,PRS)的保守、多功能接合结构域,GYF结构域蛋白在蛋白蛋白相互作用、翻译起始、mRNA剪接、监管与翻译抑制、泛素连接、信号转导、免疫蛋白酶调控等方面起重要作用。本文从结构域特性、与靶蛋白的相互作用及其功能等方面综述GYF结构域蛋白的研究进展。     

Direct production of proteins with N-terminal cysteine for site-specific conjugation

Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

Sequence-structure-function relation characterized in silico

Methods for biological function recognition in silico appeared to be useful also for identifying characteristics of structure-to-function relations. The introduction of a three-dimensional Gauss function was assumed to represent the hydrophobic core in a protein molecule. The discrepancy between idealized "fuzzy oil-drop" and the observed one in real proteins appeared to be localized in the ligation site or in the area of biological function related part of protein molecule. The examples of proteins presented in this paper reveal that the structure-function relation can be evaluated and characterized also using the profile of the difference in value between idealized and real hydrophobicity distribution along the polypeptide chain. The specificity of particular polypeptide chain fragments in respect to their biological function and their specific participation in active site creation is discussed in this paper. The scale allowing comparison of different proteins in respect to their ligand-binding sites characteristics is introduced.     

Enhancing native chemical ligation for challenging chemical protein syntheses

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.     

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

Studies of ion channels using expressed protein ligation

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review.     

Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening

Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.     

Recent advances in the application of expressed protein ligation to protein engineering

Expressed protein ligation is a technique for joining recombinantly expressed proteins to polypeptides containing biophysical probes, post-translational modifications or unnatural amino acids. Recent advances have expanded the scope of expressed protein ligation and have allowed the approach to be applied to the study of basic biological questions.