Cytoplasmic Expression System Research Articles

The Application of FOXO1A Expression Predicts Aggressive Behavior and Poor Prognosis in Gliomas.

Forkhead box, class O, 1A (FOXO1A) is an important factor related to some human malignancies. We tested the association between FOXO1A cytoplasmic expression and World Health Organization grading system in primary brain tumors (PBTs). First of all, Western blot analysis was also performed in normal brain tissue and U87MG, LN229, GBM8401, and U118MG glioma cell lines protein lysates. Then, in order to realize FOXO1A gene expression in gliomas, U87MG, LN229, GBM8401 mRNA were applied to performed quantitative reverse transcription polymerase chain reaction (RT-PCR). At last, the immunohistochemical (IHC) analysis of FOXO1A was performed in 8 non-neoplastic brain tissues and 126 PBTs. The immunostain scores were obtained as the degree of cytoplasmic FOXO1A intensity multiplied by the percentage of positively stained tumor area. On the basis of the results of these in vitro studies, marked increase FOXO1A protein and mRNA expressions in glioma cell lines than in normal human tissue. On the view point of IHC stains, the average immunostain score of FOXO1A in all PBTs was significantly higher than non-neoplastic brain tissues. In addition, the immunostain scores of FOXO1A in high grade were higher than low-grade gliomas. Furthermore, higher cytoplasmic expression of FOXO1A might indicate the shorter overall survival rate in gliomas. Furthermore, FOXO1A expression was associated with isocitrate dehydrogenase I /2, ATRX, and p53 mutation by IHC staining. Therefore, the application of FOXO1A IHC stain might have some benefits to improve diagnostic accuracy, predict tumor progression, and evaluate survival time in glioma patients.

C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system.

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.

SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm

Antibody-enzyme fusion proteins have been used for various immunological detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chemical conjugation methods, as they require no complex chemical reactions and allow for the strict control of the number of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic soluble expression of an antigen-binding fragment (Fab) fused with Escherichia coli alkaline phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the association of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E.coli oxidative cytoplasm in soluble form having both antigen-binding and AP activity, and was purified to homogeneity by two step column chromatography, suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and solubility of the Fab-enzyme fusion in an E.coli cytoplasmic expression system.

Applying DDX3X Biomarker to Discriminate Atypical from Benign Meningiomas in Tissue Microarray.

Patients with atypical meningiomas have a higher recurrence rate and poorer prognosis than patients with benign meningeal tumors. However, differentiating atypical from benign meningiomas based on fragmented specimens from brain tumor biopsy is complicated. We tested the association of DDX3X cytoplasmic expression and World Health Organization grading system in various subtypes of meningiomas. In our study, DDX3X expression was evaluated immunohistochemically in 10 non-neoplastic brain tissues and 71 meningiomas. The immunostaining scores were calculated as the product of cytoplasmic DDX3X intensity and the percentage of positively stained cells. Our results revealed most of the non-neoplastic brain tissues were immunonegative for DDX3X. The average DDX3X immunostaining score was significantly higher in meningiomas than non-neoplastic brain tissues and significantly higher in atypical meningiomas than in various subtypes of benign meningiomas. In conclusion, DDX3X immunohistochemistry combined with hematoxylin and eosin staining may help differentiate atypical meningiomas from benign meningeal tumors.

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli.

Background:Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process.Objectives:In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b).Materials and Methods:The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd.Results:Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band.Conclusions:The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step.

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37-nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia-based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small-scale expression in 10(6) cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ∼39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.

Evaluating the conformation of recombinant domain I of β2-glycoprotein I and its interaction with human monoclonal antibodies

Pathogenic antiphospholipid antibodies (aPL) cause the antiphospholipid syndrome (APS) by interacting with domain I (DI) of beta-2-glycoprotein I (β2GPI). The aPL/β2GPI complex then exerts pathogenic effects on target cells. We previously described periplasmic bacterial expression of native and mutated variants of DI, and reported the presence of immunodominant epitopes at positions 8–9 (D8/D9) and position 39 (R39). Mutations at these positions strongly influenced the ability of recombinant DI to bind patient-derived IgG aPL and to inhibit pathogenic effects of these aPL in a mouse model of APS.We now describe an improved cytoplasmic bacterial expression system allowing higher yield of DI. We demonstrate that the nuclear magnetic resonance (NMR) spectra of a 15N,13C-isotope-labelled sample of the recombinant DI protein exhibit properties consistent with the structure of DI in crystal structure of intact β2GPI. Mutations at D8/D9 and R39 had limited impact on the NMR spectrum of DI indicating maintenance of the overall fold of the DI domain.We investigated interactions between five variants of DI and ten monoclonal human IgG antibodies, all derived from the IgG aPL antibody IS4 by sequence manipulation and in vitro expression. Arginine residues at positions 100 and 100g in IS4VH CDR3 play a particularly important role in binding to DI, but this is unlikely to be due to electrostatic interactions with negatively charged amino acids on DI. Both the strength of binding to DI and the ability to discriminate different DI variants varies between the different IgG antibodies tested. There was no simple relationship between these binding properties and antibody pathogenicity.

Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b

The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9–11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1 342–770 region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG 85, similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG 58 or under certain conditions GUG 26 internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal -2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the -2/+1 and -1/+2 frameshift events operating at the HCV-1 core codons 8-11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8-11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

Immunostimulatory virotherapy using recombinant sendai virus as a new cancer therapeutic regimen

The utility of recombinant Sendai virus (rSeV) has been considerably examined over the last decade as a potent gene transfer candidate in a cytoplasmic gene expression system. Such risks as excessive immune responses associated with this virus administration in vivo however have limited its applicability in clinical settings as is the case with other viral vectors including adenoviruses. In consequence of extensive assessment on the mechanisms of immune responses against SeV, we found that ex vivo infection of immature dendritic cells (DCs) with SeV demonstrates their spontaneous maturation and activation. We applied this result to create a unique, representative, and powerful agent to activate DCs, namely rSeV-modified DCs (rSeV/DCs), for use in cancer immunotherapy. Use of this system in vivo resulted in the induction of efficient antitumor immunity against vascularized rodent tumors, including melanoma, hepatocellular carcinoma, neuroblastoma, squamous cell carcinoma, and prostatic cancer, and it even frequently associated with elimination of those tumors. These results indicate that rSeV could be a powerful immune booster for DC-based cancer immunotherapy that is worth investigating further. We propose a conceptual term "immunostimulatory virotherapy" to describe this new method of cancer therapy using the rSeV/DCs system.

Immunostimulatory virotherapy using recombinant Sendai virus as a new cancer therapeutic regimen

The utility of recombinant Sendai virus (rSeV) has been considerably examined over the last decade as a potent gene transfer candidate in a cytoplasmic gene expression system. Such risks as excessive immune responses associated with this virus administration in vivo however have limited its applicability in clinical settings as is the case with other viral vectors including adenoviruses. In consequence of extensive assessment on the mechanisms of immune responses against SeV, we found that ex vivo infection of immature dendritic cells (DCs) with SeV demonstrates their spontaneous maturation and activation. We applied this result to create a unique, representative, and powerful agent to activate DCs, namely rSeV-modified DCs (rSeV/DCs), for use in cancer immunotherapy. Use of this system in vivo resulted in the induction of efficient antitumor immunity against vascularized rodent tumors, including melanoma, hepatocellular carcinoma, neuroblastoma, squamous cell carcinoma, and prostatic cancer, and it even frequently associated with elimination of those tumors. These results indicate that rSeV could be a powerful immune booster for DC-based cancer immunotherapy that is worth investigating further. We propose a conceptual term "immunostimulatory virotherapy" to describe this new method of cancer therapy using the rSeV/DCs system.

Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells

Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.

Investigation of factors responsible for cell line cytoplasmic expression differences

BackgroundPrevious work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line) and COS-7 (a low expressing cell line).ResultsThe main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence) are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes.ConclusionAfter exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

Factors limiting autogene‐based cytoplasmic expression systems

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20-fold higher than that of a CMV nuclear expression plasmid control. Here various strategies are described to increase the levels of autogene-based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES element. Although insights into the function of various IRES elements were gained, none of these changes demonstrated a significant increase in gene expression. However, determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments revealed that transgene mRNA levels were saturated at up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production, as well as translation, are important factors limiting autogene-based cytoplasmic expression.

314. mRNA Production Is the Major Factor Limiting Autogene-Based Cytoplasmic Expression

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20 fold higher than that of a CMV nuclear expression plasmid control. Here we describe various strategies to increase the levels of autogene based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES sequence. It was found that none of these changes demonstrated a significant increase in gene expression. However determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments, revealed transgene mRNA levels up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production is a major factor limiting autogene based cytoplasmic expression.

722. SeV RNA Replicon Vector: An Efficient Cytoplasmic Expression System Derived from Sendai Virus Self-Replicating Ribonucleoprotein Complexes

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative strand RNA belonging to Paramyxoviridae family. We have previously shown that recombinant SeV as well as its modified non-transmissible variants can efficiently transfer and express foreign genes for a broad range of mammalian cells and tissues in vitro and in vivo and are suitable for use in gene therapy. SeV has two envelope glycoproteins HN and F, which mediate the virus attachment to and penetration into target cells, respectively. These envelope glycoproteins are not only important factors for viral tropism but also major determinants of immunogenicity in infected animals. Here we report the development of a novel SeV RNA replicon vector system that takes advantage of SeV self-replicating ribonucleoprotein complexes (RNP). In the vector system, RNP itself is non-infectious and can be infectious by the addition of lipofection reagents. In this system, nuclear entry of SeV RNA genome which forms an RNP complex with SeV NP, P and L proteins is not required and high expression of transgene is expected in transduced cells by self-replication of RNP in the cytoplasm. In addition, no effect of transduction of transgene in the presence of neutral antibody against SeV is expected. As the source of RNP, non-infectious virus-like particles (VLP) were recovered by infecting F-defective SeV vector into LLC-MK2 cells and harvesting VLP from the supernatants of the infected cells. To qualitatively and quantitatively compare this new vector system with conventional SeV vector or plasmid DNA, we introduced β-galactosidase, luciferase or enhanced green fluorescent protein (EGFP) reporter gene into the F-defective SeV genome, and recovered VLP from cloned cDNAs. The SeV RNA replicon vector mixed with lipofection reagents efficiently transferred foreign genes to several mammalian cell lines in vitro, and dose-dependently expressed luciferase or EGFP genes in tranduced cells. The expression level of SeV RNA replicon vector was comparable to that of SeV F-defective vector and remarkably higher than plasmid DNA-liposome complexes. Transduction of a foreign gene by SeV RNA replicon vector was not inhibited by the addition of an anti-SeV-HN antibody, although transduction by conventional SeV vector was completely inhibited. Therefore, the SeV RNA replicon vector system is useful for cytoplasmic expression of foreign genes in vitro and may be of help to its application to human gene therapy such as repeated administration and targeting of the vector.

An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression.

The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.

Optimization of the Antibody C H3 Domain by Residue Frequency Analysis of IgG Sequences

In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 C H3 domains and examined their folding/refolding characteristics. We utilized a cytoplasmic bacterial expression system with a thioredoxin reductase knock-out strain of BL21(DE3) to produce bovine, murine and human C H3. Under identical conditions, expression of bovine C H3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C H3 have surprisingly similar structures and stabilities, considering the marginal sequence conservation between the two molecules. Residue frequency analysis using a limited data set of 36 unique Fc sequences originating from 19 different mammalian species targeted five specific sites for optimization within bovine C H3. Combination of three of these mutants increased the thermal stability of the molecule to 86 °C. Comparison of this approach to similar studies using larger sequence databases and/or different selection criteria suggests sequence database design can increase the success rate for identifying residue sites worth optimizing. This optimized C H3 domain can be used as a particularly stable platform for functional design and can be grafted into full-length antibody sequences to enhance their thermodynamic parameters and shelf-life.