Factors and Mechanisms Affecting the Secretion of Recombinant Protein in CHO Cells.

The market demand for recombinant therapeutic proteins (RTPs) has promoted the development of various protein expression host and bioprocessing technologies. Since mammalian cells have the unique advantage of being able to direct the correct folding of proteins and provide post-translational processing such as complex glycosylation, the RTPs produced by them currently account for approximately 80% of the approved marketed RTPs. Among them, Chinese hamster ovary (CHO) cells are currently the preferred host cells for the production of RTPs. Production of RTPs in CHO cells involves the synthesis, processing, transport, and secretion of proteins. The secretion process of proteins is one of the key steps, which greatly limits the yield and quality of RTPs. Here, we review the recombinant protein secretion process of CHO cells and its influencing factors, and further discuss the optimization strategy for recombinant protein secretion and expression in CHO cells.

Kineococcus glutineturens sp. nov., isolated from soil in Yunnan, south-west China.

An orange-coloured, non-spore-forming, motile and coccus-shaped actinobacterium, designated YIM 75677(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan Province, south-west China and its taxonomic position was investigated. Growth of strain YIM 75677(T) occurred at 12-55 °C, pH 6.0-9.0 and NaCl tolerance up to 2 % (w/v). Cells adhered to agar media and were agglutinated tightly together. The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose, mannose and ribose. The predominant menaquinone was MK-9 (H(2)) and the major fatty acids were anteiso-C(15:0) and iso-C(15:0). Mycolic acids were not present. The DNA G+C content of strain YIM 75677(T) was 74.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons clearly revealed that strain YIM 75677(T) represents a novel member of the genus Kineococcus and is closely related to Kineococcus xinjiangensis S2-20(T) (level of similarity, 98.6 %). Meanwhile, the result of DNA-DNA hybridization between strain YIM 75677(T) and K. xinjiangensis S2-20(T) demonstrated that this isolate represented a different genomic species in the genus Kineococcus. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 75677(T) represents a novel species of the genus Kineococcus, for which the name Kineococcus glutineturens sp. nov. is proposed. The type strain is YIM 75677(T) (=CCTCC AA 209075(T) = JCM 18126(T)).

Paenibacillus frigoriresistens sp. nov., a novel psychrotroph isolated from a peat bog in Heilongjiang, Northern China.

A novel cold-resistant bacterium, designated YIM 016(T), was isolated from a peat bog sample collected from Mohe County, Heilongjiang Province, Northern China and its taxonomic position was investigated using a polyphasic approach. The strain was Gram-positive, aerobic, endospore-forming, motile and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequence clearly revealed that strain YIM 016(T) is a member of the genus Paenibacillus. The strain is closely related to Paenibacillus alginolyticus DSM 5050(T), Paenibacillus chondroitinus DSM 5051(T) and Paenibacillus pocheonensis Gsoil 1138(T) with similarities of 99.0 %, 97.0 % and 96.3 %, respectively. Meanwhile, the low DNA-DNA relatedness levels between strain YIM 016(T) and its closely related phylogenetic neighbours demonstrated that this isolate represents a new genomic species in the genus Paenibacillus. Phenotypic and chemotaxonomic tests showed that growth of strain YIM 016(T) occurred at 4-37 °C, pH 6.0-8.0 and with a NaCl tolerance up to 0.5 % (w/v). The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose and ribose. The predominant menaquinone was MK-7 and the major fatty acids were anteiso-C(15:0) and iso-C(16:0). The DNA G+C content of strain YIM 016(T) was 51.7 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 016(T) could be clearly distinguished from other species of the genus Paenibacillus. It is therefore concluded that strain YIM 016(T) represents a novel species in the genus Paenibacillus, for which the name Paenibacillus frigoriresistens sp. nov. is proposed. The type strain is YIM 016(T) (= CCTCC AB 2011150(T) = JCM 18141(T)).

Geodermatophilus nigrescens sp. nov., isolated from a dry-hot valley.

A novel actinomycete, designated as strain YIM 75980(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan province, south-west China and was subjected to polyphasic taxonomic characterization. The organism produced circular, smooth, red to black coloured colonies comprising coccoid-shaped cells. Colonies on agar medium lacked mycelia and cells adhered to the agar. Strain YIM 75980(T) contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and contained galactose, arabinose and glucosamine as the main sugars in the whole-cell hydrolysates. The predominant menaquinone was MK-9 (H(4)) and the major fatty acids were iso-C(15:0), iso-C(16:0) and C(16:0). The DNA G + C content of strain YIM 75980(T) was 73.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences clearly showed that strain YIM 75980(T) formed a distinct clade within the genus Geodermatophilus and was closely related to Geodermatophilus obscurus DSM 43160(T) (level of similarity, 97.9%). Furthermore, the result of DNA-DNA hybridization between strain YIM 75980(T) and G. obscurus 43160(T) demonstrated that this isolate represented a different genomic species in the genus Geodermatophilus. Moreover, the physiological and biochemical data showed the differentiation of strain YIM 75980(T) from its closest phylogenetic neighbour. Therefore, it is proposed that strain YIM 75980(T) represents a novel species of the genus Geodermatophilus, for which the name Geodermatophilus nigrescens sp. nov. is proposed. The type strain is YIM 75980(T) (=CCTCC AA 2011015(T) =JCM 18056(T)).

Amycolatopsis dongchuanensis sp. nov., an actinobacterium isolated from soil.

A novel actinomycete strain, designated YIM 75904(T), was isolated from a soil sample that had been collected from a dry and hot river valley in Dongchuan county, Yunnan province, south-western China. The taxonomic position of the novel strain was investigated by a polyphasic approach. In phylogenetic analyses based on 16S rRNA gene sequences, strain YIM 75904(T) formed a distinct clade within the genus Amycolatopsis and appeared to be closely related to Amycolatopsis sacchari K24(T) (99.3% sequence similarity). Strain YIM 75904(T) had a type-IV cell wall, with no detectable mycolic acids, and had MK-9(H(4)) as its predominant menaquonine. Its cell wall contained meso-diaminopimelic acid, galactose, glucose and arabinose, and its major cellular fatty acids were iso-C(16:0), iso-C(15:0), anteiso-C(17:0) and anteiso-C(15:0). The genomic DNA G+C content of the novel strain was 68.5 mol%. Based on the results of physiological and biochemical tests and DNA-DNA hybridizations, strain YIM 75904(T) represents a novel species of the genus Amycolatopsis for which the name Amycolatopsis dongchuanensis sp. nov. is proposed. The type strain is YIM 75904(T) (=CCTCC AA 2011016(T) =JCM 18054(T)).

[Mutation analysis of the pathogenic gene in a Chinese family with Brachydactyly type B1].

We identified and characterized a Chinese family with autosomal dominant Brachydactyly type B1 (BDB1). Linkage analysis revealed that the disease gene of the Chinese BDB1 family was linked to ROR2 locus. Mutational hot spot of ROR2 gene was amplified by polymerase chain reaction (PCR) and sequenced directly. A c.2265C>A heterozygous mutation was detected in all of the patients. This mutation led to the change of p.Y755X in protein level and a truncated ROR2 protein losing integrant domains was generated. The mutation was detected in all the patients, but not in all the normal individuals of this family and 50 normal controls. This paper for the first time reported a c.2265C>A mutation in ROR2 gene of a family with BDB1 in China, which enriches ROR2 gene mutation spectrum in Chinese with BDB1.

[The mutation spectrum of phenylalanine hydroxylase gene in patients with phenylketonuria in Henan province].

OBJECTIVE: To investigate the characteristics of the phenylalanine hydroxylase (PAH) gene mutations in patients with phenylketonuria (PKU) in Henan province, China, in order for providing basic information for clinical genetic counseling and prenatal diagnosis. METHODS: All the exons and partial flanking introns of the PAH gene were detected by polymerase chain reaction (PCR) and bi-directional sequencing in 34 patients with PKU from Henan province. RESULTS: A total of 23 different disease-causing mutations were identified which corresponded to 92.65% (63/68) of the PAH alleles, including 12 missense mutations, 4 nonsense mutations, 4 splicing junction mutations, and 3 deletion mutations. Among them, A156P and P69_S70delinsP(delCTT) were novel mutations; IVS2+ 5G to C, G332E, IVS10-14C to G and L367 to Wfs were reported in Chinese population for the first time according to the PAH database (www.pahdb.mcgill.ca). Among all the 13 exons, exon 7 harbored the most type of mutations, exon 11 and exon 5 the second. The most common mutations included R243Q (17.65%, 12/68), V399V (11.76%, 8/68), IVS4-1G to A (8.82%, 6/68), R400T(7.35%, 5/68), Y166X(5.88%,4/68) and G247R(5.88%, 4/68). In addition, 9 other gene variations were found in this study. CONCLUSION: The mutation spectrum and frequency of the PAH gene of patients with phenylketonuria in Henan province were slightly different from those from other parts of China.