Effects of genome instability of parental CHO cell clones on chromosome number distribution and recombinant protein production in parent-derived subclones.

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability.

A scalable overexpression of a thermostable recombinant poly-histidine tag carboxyl esterase under lambda promoter: purification, characterization, and protein modelling.

BACKGROUND: As a white biotechnological trend, esterases are thought to be among the most active enzymes' classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants. RESULTS: A poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8-10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis-Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min-1), and (2.619 mol/ min), respectively. CONCLUSION: Creation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

The role of antifreeze genes in the tolerance of cold stress in the Nile tilapia (Oreochromis niloticus).

BACKGROUND: Tilapia is one of the most essential farmed fishes in the world. It is a tropical and subtropical freshwater fish well adapted to warm water but sensitive to cold weather. Extreme cold weather could cause severe stress and mass mortalities in tilapia. The present study was carried out to investigate the effects of cold stress on the up-regulation of antifreeze protein (AFP) genes in Nile tilapia (Oreochromis niloticus). Two treatment groups of fish were investigated (5 replicates of 15 fish for each group in fibreglass tanks/70 L each): 1) a control group; the fish were acclimated to lab conditions for two weeks and the water temperature was maintained at 25 °C during the whole experimental period with feeding on a commercial diet (30% crude protein). 2) Cold stress group; the same conditions as the control group except for the temperature. Initially, the temperature was decreased by one degree every 12 h. The fish started showing death symptoms when the water temperature reached 6-8 °C. In this stage the tissue (muscle) samples were taken from both groups. The immune response of fish exposed to cold stress was detected and characterized using Differential Display-PCR (DD-PCR). RESULTS: The results indicated that nine different up-regulation genes were detected in the cold-stressed fish compared to the control group. These genes are Integrin-alpha-2 (ITGA-2), Gap junction gamma-1 protein-like (GJC1), WD repeat-containing protein 59 isoform X2 (WDRP59), NUAK family SNF1-like kinase, G-protein coupled receptor-176 (GPR-176), Actin cytoskeleton-regulatory complex protein pan1-like (PAN-1), Whirlin protein (WHRN), Suppressor of tumorigenicity 7 protein isoform X2 (ST7P) and ATP-binding cassette sub-family A member 1-like isoform X2 (ABCA1). The antifreeze gene type-II amplification using a specific PCR product of 600 bp, followed by cloning and sequencing analysis revealed that the identified gene is antifreeze type-II, with similarity ranging from 70 to 95%. The in-vitro transcribed gene induced an antifreeze protein with a molecular size of 22 kDa. The antifreeze gene, ITGA-2 and the WD repeat protein belong to the lectin family (sugar-protein). CONCLUSIONS: In conclusion, under cold stress, Nile tilapia express many defence genes, an antifreeze gene consisting of one open reading frame of approximately 0.6 kbp.

Molecular Cloning Using In Vivo DNA Assembly.

Here we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues.

HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection.

HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for an accurate interpretation of in vitro data. However, variations in the HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell-cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than four-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated the subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

Monoclonal Antibodies Generation: Updates and Protocols on Hybridoma Technology.

Since its inception in 1975, the hybridoma technology revolutionized science and medicine, facilitating discoveries in almost any field from the laboratory to the clinic. Many technological advancements have been developed since then, to create these "magical bullets." Phage and yeast display libraries expressing the variable heavy and light domains of antibodies, single B-cell cloning from immunized animals of different species including humans or in silico approaches, all have rendered a myriad of newly developed antibodies or improved design of existing ones. However, still the majority of these antibodies or their recombinant versions are from hybridoma origin, a preferred methodology that trespass species barriers, due to the preservation of the natural functions of immune cells in producing the humoral response: antigen specific immunoglobulins. Remarkably, this methodology can be reproduced in small laboratories without the need of sophisticate equipment. In this chapter, we will describe the most recent methods utilized by our Monoclonal Antibodies Core Facility at the University of Texas-M.D. Anderson Cancer Center. During the last 10 years, the methods, techniques, and expertise implemented in our core had generated more than 350 antibodies for various applications.

Genetic analysis of Curcuma species from Asia based on intron regions of genes encoding diketide-CoA synthase and curcumin synthase.

Intron length polymorphism (ILP) markers in genes encoding diketide-CoA synthase (DCS) and curcumin synthase (CURS) showed high identification rates in 13 Curcuma species from Asia. However, the sequences of the intron regions have not yet been analyzed. To elucidate the sequence differences in intron regions of the DCS and CURS genes and to search for specific sequences suitable for the identification of Curcuma species, a large number of sequences were determined through subcloning coupled with sequencing analysis of six Curcuma plant specimens belonging to five species that showed distinct ILP patterns. More than 30 sequences of each region from each specimen were grouped into genes DCS1, DCS2, or CURS1-3 and subsequently the sequences of the same genes were compared. Sequences belonging to the same gene showed inter-species similarity, and thus, these intron sequences were less informative within each single-gene region. The determined sequences from each specimen showed 3-5 kinds of sequence lengths in DCS intron I region, and 5-7 kinds of sequence lengths in CURS intron region. The length of determined sequences and the fragment number in each intron region were different among species, or specimens in C. longa, which were in accordance with the fragment lengths and numbers in their corresponding ILP patterns.

Analysis of whole-exome data of cfDNA and the tumor tissue of non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) has the highest cancer mortality rate in the world, but currently there is no effective method of dynamic monitoring. Gene mutation is an important factor in tumorigenesis and can be detected using high-throughput sequencing technology. This study aimed to analyze the driving genes in the tumor of NSCLC patients by whole exon sequencing, and to compare and analyze the subclones of the tumor at different time points. METHODS: We collected 87 cases of NSCLC tumor tissues, para-cancer tissues, and peripheral blood samples for detecting cell-free DNAs (cfDNAs) from January 2016 to December 2018, and whole-exome sequencing was performed. The gene mutation map of NSCLC was drawn in detail by second-generation sequencing data analysis and new driver genes were found. In addition, we performed a subclonal analysis of tumors from different stages of the same patient to further describe the tumor heterogeneity. RESULTS: We found that the clonal analysis obtained by cfDNA detection was similar to the clonal analysis of the tissue samples, so real-time monitoring of tumor changes can be carried out through monitoring cfDNA. CONCLUSIONS: This study provides evidence for studying the gene mutation information of NSCLC and shows the importance of cfDNA in the analysis of tumor subcloning information.

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea.

BACKGROUND: Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. METHODS AND RESULTS: The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. CONCLUSIONS: This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

Subcloning induces changes in the DNA-methylation pattern of outgrowing Chinese hamster ovary cell colonies.

Chinese hamster ovary (CHO) cells are the most extensively used mammalian production system for biologics intended for use in humans. A critical step in the establishment of production cell lines is single cell cloning, with the objective of achieving high productivity and product quality. Despite general use, knowledge of the effects of this process is limited. Importantly, single cell cloned cells display a wide array of observed phenotypes, which so far was attributed to the instability and variability of the CHO genome. In this study we present data indicating that the emergence of diverse phenotypes during single cell cloning is associated with changes in DNA methylation patterns and transcriptomes that occur during the subcloning process. The DNA methylation pattern of each analyzed subclone, randomly picked from all outgrowing clones of the experiment, had unique changes preferentially found in regulatory regions of the genome such as enhancers, and de-enriched in actively transcribed sequences (not including the respective promoters), indicating that these changes resulted in adaptations of the relative gene expression pattern. The transcriptome of each subclone also had a significant number of individual changes. These results indicate that epigenetic regulation is a hidden, but important player in cell line development with a major role in the establishment of high performing clones with improved characteristics for bioprocessing.

Methods for Automated Single Cell Isolation and Sub-Cloning of Human Pluripotent Stem Cells.

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell-derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications. © 2020 The Authors. Basic Protocol: Efficient automated hPSC single cell seeding and clonal expansion using the iotaSciences IsoCell platform Alternate Protocol 1: hPSC single cell seeding and clonal expansion using the Cellenion CellenONE single-cell dispenser Alternate Protocol 2: hPSC single cell seeding and clonal expansion using the Cytena single-cell dispenser Support Protocol 1: Coating cell culture plates with Geltrex Support Protocol 2: hPSC maintenance in defined feeder-free conditions Support Protocol 3: hPSC passaging in clumps Support Protocol 4: Laminin 521 coating of IsoCell plates and 96-well/384-well plates Support Protocol 5: Preparation of medium containing anti-apoptotic small molecules Support Protocol 6: 96- and 384-well target plate preparation prior to single cell seeding Support Protocol 7: Single cell dissociation of hPSCs Support Protocol 8: IsoCell-, CellenONE-, and Cytena-derived hPSC clone subculture and expansion.

Directed evolution approach to enhance efficiency and speed of outgrowth during single cell subcloning of Chinese Hamster Ovary cells.

Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Optimization of overlap extension PCR for efficient transgene construction.

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

In-Situ Gel-Free Plasmid Reassembling for Rapid Gene Subcloning and Truncation

Abstract Gene subcloning, a process in which the nucleotide sequence of interest is excised from on plasmid and inserted into another, seems to be an easy task to done. However, not all subcloning attempts are successful, even when the insert sequence and the double digested target plasmid are successfully purified form agarose gel and thought to be ready for subsequent ligation. In the current study we introduce a reliable, easy, and time consuming method for gene subcloning and also truncation. The stages are all carried out in a single microtube without any running on a gel, making it possible to accomplish a successful gene subcloning or truncation even with low concentrations of DNA molecules. Summarily, subcloning is achieved by mixing the plasmids of interest in a microtube and subjecting to restriction enzymes whose restriction sites flank the segment that is going to be subcloned. Digestion mixture is precipitated in the same microtube using isopropanol and the resultant DNA molecules are allowed to take part in a ligation reaction. The recombinant plasmids of interest are screened by colony PCR. Truncation is achieved by double- digestion of the plasmid of interest using a restriction enzyme whose restriction site flanks the segment that is going to be cut out.

Galactofuranosidase from JHA 19 Streptomyces sp.: subcloning and biochemical characterization.

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP ß-d-Galf, giving a KM value of 250 µM, and the highest enzymatic efficiency ever observed of 14 mM-1  s-1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.

A new hepatoma cell line exhibiting high susceptibility to hepatitis B virus infection.

Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

An in vitro and in silico identification of antibiofilm small molecules from seawater metaclone SWMC166 against Vibrio cholerae O1.

This study aimed to determine the antibiofilm activity of seawater microbes against Vibrio cholerae (VCO1) through functional metagenomics approach. A metagenomic library was constructed from Palk Bay seawater and the library was screened to identify the biofilm inhibitory metaclone. Metaclone SWMC166 (harbouring ∼30 kb metagenomic insert) was found to exhibit antibiofilm activity against VCO1. The biofilm inhibitory potential of partially purified ethyl acetate extract of SWMC166 (EA166) was further evaluated through microscopic studies and biochemical assays. Further, EA166 treated VCO1 divulged up-regulation of genes involved in high cell density-mediated quorum sensing (QS) pathway which was analysed by real-time PCR. In order to identify the genes of interest (within ∼30 kb insert), subcloning was performed through shotgun approach. Small molecules from positive subclones SC5 and SC8 were identified through HRLC-MS analysis. Resulted small molecules were docked against QS receptors of V. cholerae to identify the bioactive metabolites. Docking studies revealed that totally seven metabolites were able to interact with QS receptors that can possibly trigger the QS cascade and sequentially inhibit the biofilm formation and virulence factors of VCO1.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines.

Assurance of monoclonality of recombinant cell lines is a critical issue to gain regulatory approval in biological license application (BLA). Some of the requirements of regulatory agencies are the use of proper documentations and appropriate statistical analysis to demonstrate monoclonality. In some cases, one round may be sufficient to demonstrate monoclonality. In this article, we propose the use of confidence intervals for assessing monoclonality for limiting dilution cloning in the generation of recombinant manufacturing cell lines based on a single round. The use of confidence intervals instead of point estimates allow practitioners to account for the uncertainty present in the data when assessing whether an estimated level of monoclonality is consistent with regulatory requirements. In other cases, one round may not be sufficient and two consecutive rounds are required to assess monoclonality. When two consecutive subclonings are required, we improved the present methodology by reducing the infinite series proposed by Coller and Coller (Hybridoma 1983;2:91-96) to a simpler series. The proposed simpler series provides more accurate and reliable results. It also reduces the level of computation and can be easily implemented in any spreadsheet program like Microsoft Excel. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1061-1068, 2016.

A novel multigene cloning method for the production of a motile ATPase.

With the advent of nanotechnology, new functional modules (e.g., nanomotors, nanoprobes) have become essential in several medical fields. Generally, mechanical modulators systems are the principal components of most cutting-edge technologies in modern biomedical applications. However, the in vivo use of motile probes has raised many concerns due to their low sensitivity and non-biocompatibility. As an alternative, biological enzymatic engines have received increased attention. In particular, ATPases, which belong to a class of motile enzymes that catalyze chemical metabolic reactions, have emerged as a promising motor due to their improved biocompatibility and performance. However, ATPases usually suffer from lower functional activity and are difficult to express recombinantly in bacteria relative to their conventional and synthetic competitors. Here, we report a novel functional modified ATPase with both a simple purification protocol and enhanced motile activity. For this mutant ATPase, a new bacterial subcloning method was established. The ATPase-encoding sequence was redesigned so that the mutant ATPase could be easily produced in an Escherichia coli system. The modified thermophilic F1-ATPase (mTF1-ATPase) demonstrated 17.8unit/mg ATPase activity. We propose that derivatives of our ATPase may enable the development of novel in vitro and in vivo synthetic medical diagnostics, as well as therapeutics.