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Nivolumab, a fully human IgG4 anti-programed cell death 1（PD-1）antibody, is recently one of the most popular and successful therapeutic monoclonal antibodies in clinical use. With the increasing demands for Nivolumab and other therapeutic monoclonal antibodies, the mammary gland bioreactor has been regarded as another choice for the production of recombinant monoclonal antibodies besides mammalian cell culture. Here, we expressed a recombinant human anti-PD-1 antibody in the mammary glands of transgenic mice. Two expression vectors were constructed bearing the heavy and light chains of anti-PD-1 antibody respectively under the control of bovine αs1-casein promoter. Transgenic mice were then generated by co-microinjection of the two expression cassettes. Three F0 founders with both heavy chain and light chain positive were obtained. Transgenes of both chains were detected to be stably transmitted to the offspring. The recombinant antibody was detected in the milk of transgenic mice with the highest expression level up to 80.52 ± 0.82 mg/L and could specifically binds to the human PD-1 antigen. Therefore, our results suggest the feasibility of anti-PD-1 antibody production in the milk of transgenic animals. [ABSTRACT FROM AUTHOR]

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed. • Using 1/2BSM achieved more production and biomass compared with BSM. • Using 1/2BSM achieved the highest IP yield that had been reported to date. • The structure of product had been proved to be correct. [ABSTRACT FROM AUTHOR]

Let |$A$| be an |$AH$| algebra, that is, |$A$| is the inductive limit |$C^{*}$| -algebra of   A 1 → ϕ 1, 2 A 2 → ϕ 2, 3 A 3 ⟶ ⋯ ⟶ A n ⟶ ⋯ with |$A_{n}=\bigoplus_{i=1}^{t_{n}}P_{n,i}M_{[n,i]}(C(X_{n,i}))P_{n,i}$|⁠, where |$X_{n,i}$| are compact metric spaces, |$t_{n}$| and |$[n,i]$| are positive integers, and |$P_{n,i}\in M_{[n,i]}(C(X_{n,i}))$| are projections. Suppose that |$A$| has the ideal property: each closed two-sided ideal of |$A$| is generated by the projections inside the ideal, as a closed two-sided ideal. Suppose that |$\sup_{n,i}\dim(X_{n,i})<+\infty$|⁠. In this article, we prove that |$A$| can be written as the inductive limit of   B 1 ⟶ B 2 ⟶ ⋯ ⟶ B n ⟶ ⋯, where |$B_{n}=\bigoplus_{i=1}^{s_{n}}Q_{n,i}M_{\{n,i\}}(C(Y_{n,i}))Q_{n,i}$|⁠, where |$Y_{n,i}$| are |$\{pt\}, [0,1], S^{1}, T_{II, k}, T_{III, k}$| and |$S^{2}$| (all of them are connected simplicial complexes of dimension at most three), |$s_{n}$| and |$\{n,i\}$| are positive integers and |$Q_{n,i}\in M_{\{n,i\}}(C(Y_{n,i}))$| are projections. This theorem unifies and generalizes the reduction theorem for real rank zero |$AH$| algebras due to Dadarlat and Gong [ 4, 6, 17 ] and the reduction theorem for simple |$AH$| algebras due to Gong (see [ 19 ]). [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris . A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418. After optimizing the induction conditions and additional supplements, the highest yield of HSA reached 1.6 g/L in a shake flask. Performing high density fermentation further improved the highest yield to 8.86 g/L in a fermenter after 96 h of methanol induction. This result is more promising than the previous reports of industrial applications, which reported the highest yield as 92.29 mg/L/h, considering that the space-time yield of rHSA was doubled. In addition, the desired protein was purified by filtration and Cibacron Blue affinity chromatography, which yielded a 58% recovery of a product that had over a 96% purity. This study reveals that Pichia pastoris is an excellent system for recombinant human serum albumin expression due to its outstanding expression capacity. In addition, the high efficiency level of rHSA production lays a solid foundation for its use in industrial production. [ABSTRACT FROM AUTHOR]

Let A be a unital simple separable locally approximately subhomogeneous C*-algebra (locally ASH algebra). It is shown that A ⊗ Q can be tracially approximated by unital Elliott–Thomsen algebras with trivial K 1 -group, where Q is the universal UHF algebra. In particular, it follows that A is classifiable by the Elliott invariant if A is Jiang–Su stable. [ABSTRACT FROM AUTHOR]

Abstract: Let A be an AH algebra, that is, A is the inductive limit -algebra of with , where are compact metric spaces, and are positive integers, and are projections. Suppose that A has the ideal property: each closed two-sided ideal of A is generated by the projections inside the ideal, as a closed two-sided ideal. Suppose that . (This condition can be relaxed to a certain condition called very slow dimension growth.) In this article, we prove that if we further assume that is torsion free, then A is an approximate circle algebra (or an algebra), that is, A can be written as the inductive limit of where . One of the main technical results of this article, called the decomposition theorem, is proved for the general case, i.e., without the assumption that is torsion free. This decomposition theorem will play an essential role in the proof of a general reduction theorem, where the condition that is torsion free is dropped, in the subsequent paper Gong et al. (preprint) —of course, in that case, in addition to space , we will also need the spaces , , and , as in Gong (2002) . [Copyright &y& Elsevier]

Let X be a compact metric space and A = C(X). Suppose that B is a class of unital C[sup *]-algebras satisfying certain conditions, we prove the following: For any ∈ > O, finite set F ⊂ A, there is an integer I such that if φ, ψ : A → B(B ∈ B) are sufficiently multiplicative morphisms (e.g. when both φ and ψ are *-homomorphisms) which induce same K-theoretical maps, then there are a unitary u ∈ M[sub l+1](B) and a homomorphism σ : A → M[sub l](B) with finite dimensional image such that υ[sup *] diag(φ(ƒ),σ(ƒ))υ- diag(ψ(ƒ), σ(ƒ)) < ∈ for all &fnof ∈ F. In particular, the integer I does not depend on B, φ and ψ. This feature has important applications to the classification theory of nuclear C[sup *]-algebras. [ABSTRACT FROM AUTHOR]

Let A be a unital simple C*-algebra of real zero, stable rank one, with weakly unperforated K[sub 0(] A) and unique normalized quasi-trace τ, and let X be a compact metric space. We show that two monomorphisms &phgr;, ψ : C(X) → A are approximately unitarily equivalent if and only if &phgr; and ψ induce the same element in KL(C(X), A) and the two lineal functionals τ ∘ &phgr; and τ ∘ ψ are equal. We also show that, with an injectivity condition, an almost multiplicative morphism from C(X) into A with vanishing KK-obstacle is close to a homomorphism. [ABSTRACT FROM AUTHOR]

We present a classification theorem for separable amenable simple stably projectionless C ∗ -algebras with finite nuclear dimension whose K 0 vanish on traces which satisfy the Universal Coefficient Theorem. One of C ∗ -algebras in the class is denoted by Z 0 which has a unique tracial state, K 0 (Z 0) = Z and K 1 (Z 0) = { 0 }. Let A and B be two separable amenable simple C ∗ -algebras satisfying the UCT. We show that A ⊗ Z 0 ≅ B ⊗ Z 0 if and only if Ell (A ⊗ Z 0) = Ell (B ⊗ Z 0). A class of simple separable C ∗ -algebras which are approximately sub-homogeneous whose spectra having bounded dimension is shown to exhaust all possible Elliott invariant for C ∗ -algebras of the form A ⊗ Z 0 , where A is any finite separable simple amenable C ∗ -algebras. [ABSTRACT FROM AUTHOR]

The class of simple separable KK-contractible (KK-equivalent to { 0 }) C*-algebra s which have finite nuclear dimension is shown to be classified by the Elliott invariant. In particular, the class of C*-algebra s A ⊗ W is classifiable, where A is a simple separable C*-algebra with finite nuclear dimension and W is the simple inductive limit of Razak algebras with unique trace, which is bounded (see Razak (2002) and Jacelon (2013)). [ABSTRACT FROM AUTHOR]

While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/β constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC–MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test. [ABSTRACT FROM AUTHOR]

The red arrow indicates a Tm1 value of 62.45°C." now reads: "C, The Tm1 of protein was quantitatively detected by Uncle biologics stability screening platform. S5C, the melting temperature (Tm1) is 62.45 °C, lower than the control IgG SB 4 sb S228P whose Tm1 is 69.00 °C, indicating that PD-1 × LAG3 TiMab is relatively stable." now reads: "The thermostability was also examined with Uncle biologics stability screening platform (Unchained Labs). [Extracted from the article]

β-lactam antibiotics are widely used in clinic. Filamentous fungus Acremonium chrysogenum is an important industrial fungus for the production of CPC, one of the major precursors of β-lactam antibiotics. Although its fermentation yield has been bred significantly over the past decades, little is known regarding molecular changes between the industrial strain and the wild type strain. This limits the possibility to improve CPC production further by molecular breeding. Comparative transcriptome is a powerful tool to understand the molecular mechanisms of CPC industrial high yield producer compared to wild type. A total of 57 million clean sequencing reads with an average length of 100 bp were generated from Illumina sequencing platform. 22,878 sequences were assembled. Among the assembled unigenes, 9502 were annotated and 1989 annotated sequences were assigned to 121 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we compared the transcriptome differences between a high-yield and a wild-type strain during fermentation. A total of 4329 unigenes with significantly different transcription level were identified, among which 1737 were up-regulated and 2592 were down-regulated. 24 pathways were subsequently determined which involve glycerolipid metabolism, galactose metabolism, and pyrimidine metabolism. We also examined the transcription levels of 18 identified genes, including 11 up-regulated genes and 7 down-regulated genes using reverse transcription quantitative -PCR (RT-qPCR). The results of RT-qPCR were consistent with the Illumina sequencing. In this study, the Illumina sequencing provides the most comprehensive sequences for gene expression profile of Acremonium chrysogenum and allows de novo transcriptome assembly while lacking genome information. Comparative analysis of RNA-seq data reveals the complexity of the transcriptome in the fermentation of different yield strains. This is an important public information platform which could be used to accelerate the research to improve CPC production in Acremonium chrysogenum. [ABSTRACT FROM AUTHOR]

To improve the yield of recombinant human serum albumin (HSA) in Pichia pastoris by medium optimization and establish the related purification scheme. A simplified version of the generally used buffered glycerol complex medium (BMGY), which contained yeast extract, glycerol and potassium salts, was found to be applicable. By decreasing the salt concentration of basal salt medium (BSM) to half of the original formula further, we achieved a high yield of 17.47 g/L HSA in the supernatant within a 192 h induction, which is the highest rHSA yield ever reported as far as we know. Accompanied with a three-step purification procedure which recovered two thirds of the desired protein at high purity, our work lays a solid foundation for large-scale industrial production of HSA. Medium optimization plays a significant role in improving the yield of desired protein, lowering the production cost and helping to explore the producing strain's character. • The widely used BMGY was simplified by a large margin without decrease of the production. • The HSA yield achieved over 17 g/L with a modified BSM. • 11 g/L HSA at high purity could be recovered by a three-step purification procedure. [ABSTRACT FROM AUTHOR]

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthia-zole-2-carboxylic acid (ADT) using HPLC and H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety. [ABSTRACT FROM AUTHOR]