A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.     

Conformationally altered aortic myosin light chains

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.     

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as cholinesterase inhibitors against Alzheimer’s disease

Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat Alzheimer’s disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great importance. Herein, we report the synthesis, structure–activity relationship study and biological evaluation of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase, are evaluated for the amyloid β-protein self-aggregation inhibition and in vivo assays. The optimal compound 19, 27, and 30 (human AChE IC₅₀?=?10.2?±?1.2, 16.5?±?1.7, and 15.3?±?1.8?nM, respectively) show good performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hepatotoxicity evaluation. These compounds deserve further evaluation for the development of new therapeutic agents against AD.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

A generic algorithm for finding restriction sites within DNA sequences

This paper describes a generic algorithm for finding restrictionsites within DNA sequences. The ‘genericity’ ofthe algorithm is made possible through the use of set theory.Basic elements of DNA sequences, i.e. nucleotides (bases), arerepresented in sets, and DNA sequences, whether specific, ambiguousor even protein-coding, are represented as sequences of thosesets. The set intersection operation demonstrates its abilityto perform pattern-matching correctly on various DNA sequences.The performance analysis showed that the degree of complexityof the pattern matching is reduced from exponential to linear.An example is given to show the actual and potential restrictionsites, derived by the generic algorithm, in the DNA sequencetemplate coding for a synthetic calmodulin. Received on October 2, 1990; accepted on December 18, 1990     

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.