Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma ...membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na
-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His
, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His
and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.
Structure of the chymosin gene of Siberian roe deer (
Capreolus pygargus
) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of
...C. pygargus
was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1′ + S3′ specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (
Camelus dromedarius
) and cow (
Bos taurus
). Specific milk-clotting activity of the recombinant chymosin of
C. pygargus
was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of
C. pygargus
occupied an intermediate position between the genetically engineered analogs of
B. taurus
and
C. dromedarius
chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.
•The bs-Ab (HN-Fc-CD16) and ts-Ab (HN-Fc-IL15-CD16) were properly constructed.•In silico and computational analyses indicated proper exposure of Abs in bs/tsAbs.•The bi/tsAbs showed strong binding to ...both HN on TC-NDV and CD16 on NK cell surface.•The bi/tsAbs triggered Nk-specific cytotoxicity against TC-NDV (higher for ts-Ab)•The bi/tsAbs enhanced IFN-γ production by activated NK cells (higher for ts-Ab).
Cancer/tumor cells infected with the “avian paramyxovirus Newcastle Disease Virus (TC-NDV)” express the viral hemagglutinin-neuraminidase (HN) on the cell surface that is used as both the danger signal and anchor for bi/tri-specific antibodies (bs/tsAbs).We constructed a bs-Ab (HN-Fc-CD16) that bindsto HN and natural killer (NK)-CD16 receptor (FcgRIII)and a ts-Ab (HN-Fc-IL15-CD16) harbouring NK-activating cytokine “IL-15” within the bs-Ab.In silicoand computational predictions indicated proper exposure of both Abs in bs/tsAbs.Properbinding of thebi/tsAbstoHN on surface of TC-NDVandCD16+-cells was demonstrated by flow cytometry.The bi/tsAbstriggeredspecificcytotoxicity of NK cells againstTC-NDVand elicited substantial IFN-γproduction by activated NK cells(higher for ts-Ab) that sound promising for cancer immunotherapy purposes.
Objective
To compare the effects of human Trypsin-1 signal peptide and pro-peptide on the expression and secretion efficiency of human Interleukin-25 from mammalian cells.
Results
The signal peptide ...and combined signal peptide-pro-peptide sequence of human Trypsin-1 improved the secretion of human IL-25 from 1.7 to 3.2 µg/ml and 1.7 to 8.2 µg/ml, respectively. Deletion analysis identified the minimal Trypsin-1 derived secretion domain that maintains improved human Interleukin-25 production and secretion. The presence of Trypsin-1 pro-peptide sequence does not affect the function of secreted human Interleukin-25.
Conclusion
The Trypsin-1 signal peptide-pro-peptide sequence increased human IL-25 expression and secretion in mammalian cells by fivefold.
The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In ...this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile,
XBP1
splicing, thermal stability, and
in silico
hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody’s germline family, highlighting the fact that mAbs should be treated individually.
Interferon (IFN) is a cell-secreted cytokine possessing biological activities including antiviral functioning, immune regulation, and others. Interferon-alpha (IFN-α) mainly derives from plasmacytoid ...dendritic cells, which activate natural killer cells and regulate immune responses. IFN-α responds to the primary antiviral mechanism in the innate immune system, which can effectively cure acute infectious diseases. Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV). The clinical symptoms of PRV are as follows: reproductive dysfunction among pregnant sows and high mortality rates among piglets. These pose a severe threat to the swine industry. Related studies show that IFN-α has broad applications in preventing and treating viral diseases. Therefore, a PRV mouse model using artificial infection was established in this study to explore the pathogenic effect of IFN-α on PRV. We designed a sequence with IFN-α4 (M28623, Genbank) and cloned it on the lentiviral vector. CHO-K1 cells were infected and identified using WB and RT-PCR; a CHO-K1 cell line with a stable expression of the recombinant protein PoIFN-α was successfully constructed. H&E staining and virus titer detection were used to investigate the recombinant protein PoIFN-α’s effect on PR in BALB/c mice. The results show that the PoIFN-α has a preventive and therapeutic impact on PR. In conclusion, the recombinant protein can alleviate symptoms and reduce the replication of PRV in vivo.
Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric ...recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t½≈2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via “self-processing” viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t½ = 32 ± 1.2 h, MRT = 43 ± 2 h). 3D Fluorescent visualization and distribution of 125I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE−/− mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.
•The tetrameric form of rhBChE is shown to be an effective, long-acting bioscavenger toward organophosphorus poisoning.•The designed genetic construct and expression system facilitates large-scale production of the enzyme.•Neuron-associated BChE may significantly contribute to the protection against OP poisoning.
PDF
