Protein Engineering Approaches Research Articles

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OBJECTIVES/SPECIFIC AIMS: The off-target and organ-specific toxicities observed in cancer immunotherapy present an obstacle to T-cell-based therapeutics. A recent clinical trial underscored the need for improved methods to define TCR specificity after melanoma patients treated with TCR engineered T-cells suffered from fatal cardiovascular toxicity arising from the unpredicted recognition of a muscle-specific peptide. METHODS/STUDY POPULATION: To address this drawback to T-cell-based immunotherapies, we developed a novel protein engineering approach to define the peptide specificity of a given TCR. Here, directed evolution in a yeast display system produced a large scale peptide library, where recognition by the melanoma reactive DMF5 TCR acted as the guiding selective pressure. After this technique identified a panel of putative cross reactive peptides, sequence analysis and computational modeling followed by kinetic binding experiments and structural analysis determined the DMF5 TCR recognizes 2 distinct classes of peptides through chemically distinct mechanisms. RESULTS/ANTICIPATED RESULTS: This information led to the rational, structure-based design of additional cross reactive peptides and introduced a unique approach to screen the human proteome and identify the TCR targets which triggered undesired autoimmunity when this molecule was used in clinical trials. DISCUSSION/SIGNIFICANCE OF IMPACT: The distinct chemical nature of the 2 peptide classes suggest TCRs are more cross reactive than previously thought, presenting an obstacle to cell-based immunotherapy. Defining the peptide specificity of TCRs is of high interest to the immunology community, and will lead to improved approaches to designing engineered TCRs for cell therapy.

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants.

The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10-4 s-1 μM-1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (kcat/Km ) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4.IMPORTANCE DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Sulfolobus solfataricus Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.

Rational protein engineering approaches to further improve the keratinolytic activity and thermostability of engineered keratinase KerSMD

Keratinase has great potential to convert poultry waste into feed or fertilizers. In order to obtain excellent keratinase variants with improved thermostability and activity to degrade feather waste, we did rational protein engineering approaches. Using site-directed mutagenesis on keratinase FDD, two variants (Y94F and Y215F) showed higher extracellular keratinolytic activity and specific activity in Escherichia coli expression system. The C-terminus fusion on keratinase DDF created a new variant DDFD with the highest substrate specificity and keratinolytic activity of 6220±20U/mg. Besides, the performance of thermophilicity and theromostability of the variants was evaluated. The A218S and A218G showed more than 30% improvement of relative activity at 70°C while DDFD owned the most outstanding comprehensive performance between half-life (t1/2=146min) and keratinolytic activity. It is highly effective to obtain excellent keratinases by site-directed mutagenesis and C-terminus fusion based on model structure analysis, and it provides possibility of industrial application of new keratinases.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.

Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway.

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc.

Activity-independent screening of secreted proteins using split GFP

The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.

Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.

Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50°C and pH9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

Engineering fungal de novo fatty acid synthesis for short chain fatty acid production

Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Abstract Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β- d -glucose to d -glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg−1 and nearly 8-folds higher kcat/Km value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.

Stability-Mediated Epistasis Restricts Accessible Mutational Pathways in the Functional Evolution of Avian Hemoglobin.

If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that involve transient reductions in fitness. Here we examine the functional effects of multiple amino acid substitutions that contributed to an evolutionary transition in the oxygenation properties of avian hemoglobin (Hb). The set of causative changes included mutations at intradimer interfaces of the Hb tetramer. Replacements at such sites may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are enmeshed in networks of salt bridges and hydrogen bonds between like and unlike subunits; mutational reconfigurations of these atomic contacts can affect allosteric transitions in quaternary structure and the propensity for tetramer–dimer dissociation. We used ancestral protein resurrection in conjunction with a combinatorial protein engineering approach to synthesize genotypes representing the complete set of mutational intermediates in all possible forward pathways that connect functionally distinct ancestral and descendent genotypes. The experiments revealed that 1/2 of all possible forward pathways included mutational intermediates with aberrant functional properties because particular combinations of mutations promoted tetramer–dimer dissociation. The subset of mutational pathways with unstable intermediates may be selectively inaccessible, representing evolutionary roads not taken. The experimental results also demonstrate how epistasis for particular functional properties of proteins may be mediated indirectly by mutational effects on quaternary structural stability.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

BackgroundDroplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.ResultsFive hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).Conclusions Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Rigidifying flexible sites: An approach to improve stability of chondroitinase ABC I

The stability of chondroitin ABC lyase I (cABC I) at physiological temperature is one of the current obstacles to its clinical application. In this study, we used a protein engineering approach; rigidify flexible sites, to improve stability of cABC I. B-factor analysis showed a flexible loop at the N-terminal domain of cABC I which may be involved in its thermal instability and five residues in this region were replaced with proline. Thermal inactivation and thermal denaturation analysis revealed that Glu138Pro mutation increased half-life and Tm of enzyme, respectively. The Km values of mutated enzymes were slightly increased compared to the wild type enzyme. The results of limited proteolysis indicated that Glu138Pro mutant was more resistant against trypsinolysis and this variant was less quenched in both acrylamide and KI quenching experiments. Moreover, intrinsic fluorescence intensity of Glu138Pro variant was increased and its ANS fluorescence intensity was decreased, whereas no considerable changes were observed in the far-UV CD spectra. The structural analyses indicated compactness of structure of Glu138Pro enzyme which can be related to moderately enhanced stability of this mutant. This study demonstrated that rigidifying flexible residues can be considered as a possible approach to increase the stability of the protein.

Engineered Autonomous Human Variable Domains.

The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

Orchestrating a Symphony on a Single Conjugate: Aptamer Targeting, Gene Silencing, and Immunomodulation to Enhance Antitumor Response

Over the past decade, immunotherapy has rapidly changed the landscape of medicine, especially oncology. Checkpoint inhibitors and monoclonal antibodies capable of deploying the body’s own immune functions against diseased cells have been heralded as a soon-to-be standard of care for cancer. The excitement around immunotherapy has been accompanied by impressive results, although some are tailored only to a limited number of patients and others are accompanied by substantial toxicity profiles.1 Therefore, there are still unmet needs within the field.

Mimicking transient activation of protein kinases in living cells

Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1- and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.

When directed evolution met ancestral enzyme resurrection

The directed evolution of ancestral -resurrected- enzymes can give a new twist in protein engineering approaches towards more versatile and robust biocatalysts.

Bacterial chromate reduction: A review of important genomic, proteomic, and bioinformatic analysis

ABSTRACTToxic hexavalent chromium (Cr6+) released during various industrial and mining processes leads to serious environmental problems and health hazards. Cr6+ compounds are known to be highly toxic, mutagenic, teratogenic, and carcinogenic. Microorganisms such as bacteria employ various resistance mechanisms such as ion transport (efflux), reduction, DNA repair, and so on to overcome chromate toxicity. The genes responsible for such activity are either located in chromosome or in the plasmid of bacteria. In total of 2557 chromate resistance genes found in bacteria (2,368), eukarya (171), and archea (18) have been retrieved from the National Center for Biotechnology Information (NCBI) gene database obtained from both culture-dependent and -independent methods and were analyzed for their function, location, and diversification. Further proteomic analysis revealed a hydrophobic membrane protein such as ChrA belong to the chromate ion transporter (CHR) superfamily found to be involved in efflux of chromate ion from the cell cytoplasm conferring chromate resistance in bacteria were retrieved from the NCBI database and categorized in two groups of monodomain proteins with a sequence length of 123–234 amino acids, called bacterial short-chain CHR (bacterial SCHR), and bidomain proteins with a sequence length of 345–495 amino acids, called bacterial long-chain CHR (bacterial LCHR). Phylogenetic divergence study of 237 LCHR and 121 SCHR proteins was conducted using the neighbor-joining method in MEGA6.0 and found to be clustered into four and six clusters, respectively. Apart from CHR superfamily, various groups of oxidoreductase enzymes such as chromate reductase, nitroreductase, iron reductase, quinone reductase, hydrogenase, flavin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH)–dependent reductase showing potential toward reduction of chromate have also been identified in different microorganisms. Comparative structural analyses of seven well-studied enzymes involved in chromate reduction from Protein Data Bank database were categorized either NADPH-dependent flavin mononucleotide (FMN) reductase or FMN-dependent nitroreductase. In spite of structural diversity (e.g., tetramer/dimer, arrangements of helices/sheets/coils) all are found to be involved in chromate reduction. Chromate reductions mediated by transfer of electron from nicotinamide adenine dinucleotide to various substrates (electron acceptor such as chromate) through FMN cofactor. The anchoring amino acid residues such as Glu, Tyr, Ser, Asn, Phe and Arg interact with FMN in NADPH-dependent FMN reductases but Gly, Arg, and Ser are the interacting residues in FMN-dependent nitroreductase. Enzyme-specific domains such as PF00724, PF03358, and PF00881 are present in these enzymes. The enzymes with known structures and functions can be easily manipulated through protein engineering approach for potential applications.

A Cassette Approach for the Identification of Intein Insertion Sites.

Over the past decade split inteins have established themselves as powerful tools for protein engineering, protein semisynthesis, and protein functional control approaches. Their key advantage lies in the protein trans-splicing (PTS) reaction that enables posttranslational protein assembly from two independent, even synthetic, peptide precursors. However, since most split intein applications deal with fragmentation and modification of proteins, various issues can arise, ranging from reduced stability to impairment of protein folding. In this chapter I address how the usage of DNA encoded intein cassettes can streamline and speed up the identification of functional split intein insertion sites in novel proteins of interest (POI).

Atomic mutagenesis in ion channels with engineered stoichiometry.

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.