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Special Issue "Protein Engineering"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (15 August 2015) | Viewed by 53903

Special Issue Editor

Dr. Qiang "Shawn" Chen
E-Mail Website
Guest Editor
The Biodesign Institute and School of Life Sciences, 1001 S. McAllister Avenue, Arizona State University, Mail Zone 5401, Tempe, AZ 85287, USA
Interests: Plant made biologics (protein based therapeutics, vaccines, and diagnostics), antibody engineering, vaccine engineering, plant glycoengineering, gene expression, protein structure and , virology (flavivirus, alphavirus and filovirus) and immunology.

Special Issue Information

Dear Colleagues,

The world demand for protein-based reagents has increased tremendously. Recent progress in protein engineering through rational design and directed evolution in animal, plant, and microorganisms has created novel protein molecules with greatly enhanced functionality and utility. Advancements in high throughput technology also facilitate that pace of these proteins’ identification. These engineered proteins include biologics (such as human and animal vaccines and different monoclonal antibody variants, which act as therapeutics with enhanced efficacy and safety in preventing and treating diseases) and industry enzymes with increased catalytic efficiency and stability (or with novel substrate or cofactor specificity for biofuel production and manufacturing or for new low-cost materials).

This Special Issue invites original research and review manuscripts that report the recent developments and applications of protein engineering with respect to topics that include, but are not limited to:

  • Novel strategy, methodology, and platform development
  • Rational protein design, directed evolution, and high-throughput screening
  • Protein folding and unnatural amino acids
  • Novel engineered protein products
  • Vaccines, immunotherapeutics, monoclonal antibodies
  • Enzymes for biofuel and new material production
  • Glycoengineering and posttranslational modification
  • Novel protein engineering organism hosts
  • Extraction, recovery, and purification of engineered proteins
  • Economic impact and evaluation

Dr. Qiang "Shawn" Chen
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.


Keywords

  • novel strategy, methodology, and platform development
  • rational protein design, directed evolution, and high-throughput screening
  • protein folding and unnatural amino acids
  • novel engineered protein products
  • vaccines, immunotherapeutics, monoclonal antibodies
  • enzymes for biofuel and new material production
  • glycoengineering and posttranslational modification
  • novel protein engineering organism hosts
  • extraction, recovery, and purification of engineered proteins
  • economic impact and evaluation

Published Papers (13 papers)

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Research

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Article
Detection of Interactions between Proteins through Rotation Forest and Local Phase Quantization Descriptors
Int. J. Mol. Sci. 2016, 17(1), 21; https://doi.org/10.3390/ijms17010021 - 24 Dec 2015
Cited by 40 | Viewed by 3640
Abstract
Protein-Protein Interactions (PPIs) play a vital role in most cellular processes. Although many efforts have been devoted to detecting protein interactions by high-throughput experiments, these methods are obviously expensive and tedious. Targeting these inevitable disadvantages, this study develops a novel computational method to [...] Read more.
Protein-Protein Interactions (PPIs) play a vital role in most cellular processes. Although many efforts have been devoted to detecting protein interactions by high-throughput experiments, these methods are obviously expensive and tedious. Targeting these inevitable disadvantages, this study develops a novel computational method to predict PPIs using information on protein sequences, which is highly efficient and accurate. The improvement mainly comes from the use of the Rotation Forest (RF) classifier and the Local Phase Quantization (LPQ) descriptor from the Physicochemical Property Response (PR) Matrix of protein amino acids. When performed on three PPI datasets including Saccharomyces cerevisiae, Homo sapiens, and Helicobacter pylori, we obtained good results of average accuracies of 93.8%, 97.96%, and 89.47%, which are much better than in previous studies. Extensive validations have also been explored to evaluate the performance of the Rotation Forest ensemble classifier with the state-of-the-art Support Vector Machine classifier. These promising results indicate that the proposed method might play a complementary role for future proteomics research. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169
Int. J. Mol. Sci. 2015, 16(12), 28270-28284; https://doi.org/10.3390/ijms161226098 - 27 Nov 2015
Cited by 8 | Viewed by 3012
Abstract
Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight [...] Read more.
Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
TriFabs—Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery
Int. J. Mol. Sci. 2015, 16(11), 27497-27507; https://doi.org/10.3390/ijms161126037 - 17 Nov 2015
Cited by 15 | Viewed by 4519
Abstract
TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain [...] Read more.
TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with “knob”-mutations, and the variable region of the light chain (VL) fused to CH3 with matching “holes”. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent “in between” these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step
Int. J. Mol. Sci. 2015, 16(10), 23587-23603; https://doi.org/10.3390/ijms161023587 - 30 Sep 2015
Cited by 10 | Viewed by 3728
Abstract
Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more [...] Read more.
Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris
Int. J. Mol. Sci. 2015, 16(10), 23127-23142; https://doi.org/10.3390/ijms161023127 - 24 Sep 2015
Cited by 8 | Viewed by 3009
Abstract
Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most [...] Read more.
Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function
Int. J. Mol. Sci. 2015, 16(9), 22735-22753; https://doi.org/10.3390/ijms160922735 - 21 Sep 2015
Cited by 5 | Viewed by 3103
Abstract
Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because [...] Read more.
Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Engineering of Self-Assembled Fibronectin Matrix Protein and Its Effects on Mesenchymal Stem Cells
Int. J. Mol. Sci. 2015, 16(8), 19645-19656; https://doi.org/10.3390/ijms160819645 - 19 Aug 2015
Cited by 9 | Viewed by 3232
Abstract
Fibronectin (FN) contributes to cell adhesion, proliferation, and differentiation in various cell types. To enhance the activity of fibronectin at the sites of focal adhesion, we engineered a novel recombinant fibronectin (FNIII10) fragment connected to the peptide amphiphile sequence (PA), LLLLLLCCCGGDS. In this [...] Read more.
Fibronectin (FN) contributes to cell adhesion, proliferation, and differentiation in various cell types. To enhance the activity of fibronectin at the sites of focal adhesion, we engineered a novel recombinant fibronectin (FNIII10) fragment connected to the peptide amphiphile sequence (PA), LLLLLLCCCGGDS. In this study, the effects of FNIII10-PA on rat mesenchymal stem cells (rMSCs) were compared with those of FNIII10. FNIII10-PA showed the prominent protein adhesion activity. In addition, FNIII10-PA showed a significantly higher effect on adhesion, proliferation, and differentiation of rMSCs than FNIII10. Taken together, the FNIII10-containing self-assembled sequence enhanced rMSCs adhesion, proliferation, and differentiation. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Production and Evaluation of Virus-Like Particles Displaying Immunogenic Epitopes of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
Int. J. Mol. Sci. 2015, 16(4), 8382-8396; https://doi.org/10.3390/ijms16048382 - 14 Apr 2015
Cited by 13 | Viewed by 3986
Abstract
Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. However, the efficacy and safety of these vaccines are unsatisfactory, and [...] Read more.
Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. However, the efficacy and safety of these vaccines are unsatisfactory, and hence, there is a strong demand for the development of new PRRS universal vaccines. Virus-like particle (VLP)-based vaccines are gaining increasing acceptance compared to subunit vaccines, as they present the antigens in a more veritable conformation and are readily recognized by the immune system. Hepatitis B virus core antigen (HBcAg) has been successfully used as a carrier for more than 100 viral sequences. In this study, hybrid HBcAg VLPs were generated by fusion of the conserved protective epitopes of PRRSV and expressed in E. coli. An optimized purification protocol was developed to obtain hybrid HBcAg VLP protein from the inclusion bodies. This hybrid HBcAg VLP protein self-assembled to 23-nm VLPs that were shown to block virus infection of susceptible cells when tested on MARC 145 cells. Together with the safety of non-infectious and non-replicable VLPs and the low cost of production through E. coli fermentation, this hybrid VLP could be a promising vaccine candidate for PRRS. Full article
(This article belongs to the Special Issue Protein Engineering)
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Article
Design of an Osteoinductive Extracellular Fibronectin Matrix Protein for Bone Tissue Engineering
Int. J. Mol. Sci. 2015, 16(4), 7672-7681; https://doi.org/10.3390/ijms16047672 - 07 Apr 2015
Cited by 13 | Viewed by 3167
Abstract
Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with [...] Read more.
Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05). Full article
(This article belongs to the Special Issue Protein Engineering)
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Review

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Review
Site-Specific PEGylation of Therapeutic Proteins
Int. J. Mol. Sci. 2015, 16(10), 25831-25864; https://doi.org/10.3390/ijms161025831 - 28 Oct 2015
Cited by 174 | Viewed by 7466
Abstract
The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and [...] Read more.
The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG) group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling. Full article
(This article belongs to the Special Issue Protein Engineering)
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Review
High-Throughput Screening in Protein Engineering: Recent Advances and Future Perspectives
Int. J. Mol. Sci. 2015, 16(10), 24918-24945; https://doi.org/10.3390/ijms161024918 - 20 Oct 2015
Cited by 26 | Viewed by 5072
Abstract
Over the last three decades, protein engineering has established itself as an important tool for the development of enzymes and (therapeutic) proteins with improved characteristics. New mutagenesis techniques and computational design tools have greatly aided in the advancement of protein engineering. Yet, one [...] Read more.
Over the last three decades, protein engineering has established itself as an important tool for the development of enzymes and (therapeutic) proteins with improved characteristics. New mutagenesis techniques and computational design tools have greatly aided in the advancement of protein engineering. Yet, one of the pivotal components to further advance protein engineering strategies is the high-throughput screening of variants. Compartmentalization is one of the key features allowing miniaturization and acceleration of screening. This review focuses on novel screening technologies applied in protein engineering, highlighting flow cytometry- and microfluidics-based platforms. Full article
(This article belongs to the Special Issue Protein Engineering)
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Review
Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research
Int. J. Mol. Sci. 2015, 16(10), 23143-23164; https://doi.org/10.3390/ijms161023143 - 24 Sep 2015
Cited by 9 | Viewed by 4884
Abstract
Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific [...] Read more.
Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. Full article
(This article belongs to the Special Issue Protein Engineering)
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Review
Rational Protein Engineering Guided by Deep Mutational Scanning
Int. J. Mol. Sci. 2015, 16(9), 23094-23110; https://doi.org/10.3390/ijms160923094 - 23 Sep 2015
Cited by 10 | Viewed by 4230
Abstract
Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and [...] Read more.
Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design. Full article
(This article belongs to the Special Issue Protein Engineering)
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