Molecular cloning, recombinant protein expression, tissue distribution and functional analysis of a new c-type lysozyme from Lezhi black goat rumen

2014 ◽  
Vol 94 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Peng Zhang ◽  
Yong Wang ◽  
Mingfeng Jiang ◽  
Lianlian Zhu ◽  
Jianbo Li ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Tissue Distribution ◽  
Molecular Mass ◽  
Molecular Cloning ◽  
Recombinant Protein Expression ◽  
Black Goat ◽  
Goat Rumen

Zhang, P., Wang, Y., Jiang, M., Zhu, L., Li, J., Luo, M., Ren, H. and Liu, L. 2014. Molecular cloning, recombinant protein expression, tissue distribution and functional analysis of a new c-type lysozyme from Lezhi black goat rumen. Can. J. Anim. Sci. 94: 27–34. Three major distinct types of lysozymes have been identified in the animal kingdom and most lysozymes cloned from ruminants belong to the chicken-type (c-type). In this study, a new c-type lysozyme gene, named LZRLyz, was cloned and sequenced from the Lezhi black goat rumen. The LZRLyz cDNA has a 444 bp open reading frame (ORF) encoding a 147 amino acid polypeptide. The encoded polypeptide is predicted to have an 18 amino acid signal peptide, and a 129 amino acid mature protein with an isoelectric point (pI) of 6.08. The LZRLyz amino acid sequence shares 70.27% identity with the Capra hircus blood lysozyme and is grouped with other ruminants c-type lysozymes using the phylogenetic tree estimated by Neighbor-Jointing method. The recombinant expressed LZRLyz protein (pET-rLZR) shows a molecular mass of ∼33 kDa, which is consistent with the predicted fusion protein molecular mass and shows antimicrobial activity. Quantitative real-time RT-PCR analyses revealed that LZRLyz transcripts are expressed in all tested tissues with the predominant expression being observed in rumen and the weakest one in spleen. Results of this study suggest that the LZRLyz gene represents a new c-type lysozyme gene that likely functions in Lezhi black goat host immunity and digestive systems.

scholarly journals Influence of the second amino acid on recombinant protein expression

2010 ◽  
Vol 74 (2) ◽  
pp. 248-256 ◽  
Author(s):  
Louis Bivona ◽  
Zhongcheng Zou ◽  
Nicole Stutzman ◽  
Peter D. Sun
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression

Characterization and functional expression in yeast of a cDNA encoding NADP-dependent malic enzyme from castor oil seed

2005 ◽  
Vol 83 (2) ◽  
pp. 237-241 ◽  
Author(s):  
Heather L Shearer ◽  
David T Dennis
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Malic Enzyme ◽  
Fatty Acid Biosynthesis ◽  
Ricinus Communis ◽  
Recombinant Protein Expression ◽  
Transit Peptide ◽  
Ph Optimum ◽  
Chloroplast Transit Peptide

A cDNA encoding NADP-dependent malic enzyme was isolated from castor bean (Ricinus communis L.) endosperm. The 2362-bp cDNA encodes a 641 amino acid protein, which is predicted to have a 50 amino acid chloroplast transit peptide. Southern blotting revealed a single copy of the gene. Recombinant protein expression confirmed that the cDNA encodes a NADP-dependent malic enzyme; both Western blotting and enzyme assays detected NADP-dependent malic enzyme. Removal of the transit peptide in the recombinant protein did not affect the pH optimum of the enzyme, but it did result in an increased specific activity.Key words: Ricinus communis, NADP-dependent malic enzyme (NADP-ME), fatty acid biosynthesis, leucoplast, recombinant protein expression, transit peptide.

scholarly journals Choice of selectable marker affects recombinant protein expression in cells and exosomes

2021 ◽  
pp. 100838
Author(s):  
Chenxu Guo ◽  
Francis K. Fordjour ◽  
Shang Jui Tsai ◽  
James C. Morrell ◽  
Stephen J. Gould
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Selectable Marker ◽  
Recombinant Protein Expression ◽  
In Cells

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells

2019 ◽  
pp. 205-208 ◽  
Author(s):  
Deepak B. Thimiri Govinda Raj ◽  
Niamat Ali Khan ◽  
Srisaran Venkatachalam ◽  
Sivakumar Arumugam
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Mammalian Cells ◽  
Recombinant Protein Expression ◽  
Expression In Mammalian Cells

Evaluation of somatic embryos of alfalfa for recombinant protein expression

2014 ◽  
Vol 34 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Guohua Fu ◽  
Vojislava Grbic ◽  
Shengwu Ma ◽  
Lining Tian
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Somatic Embryos ◽  
Recombinant Protein Expression

scholarly journals Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

2015 ◽  
Vol 89 (13) ◽  
pp. 6746-6760 ◽  
Author(s):  
Nenavath Gopal Naik ◽  
Huey-Nan Wu
Keyword(s):  
Life Cycle ◽  
Recombinant Protein ◽  
Dengue Virus ◽  
Protein Expression ◽  
Nonstructural Protein ◽  
Recombinant Protein Expression ◽  
Rna Replication ◽  
Viral Production ◽  
Glycosylation Sites ◽  
Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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