An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E. coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.
Escherichia coli ; Fermentation ; Recombinant Proteins ; biosynthesis ; Sarcosine Oxidase ; biosynthesis

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.
Animals ; Biocompatible Materials ; Collagen/biosynthesis* ; Humans ; Hydroxylation ; Recombinant Proteins/biosynthesis*

To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.
Animals ; Neurotoxins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Reptilian Proteins ; biosynthesis ; genetics ; Spider Venoms ; biosynthesis ; genetics

Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.
Animals ; Arthropod Proteins ; biosynthesis ; Baculoviridae ; Bombyx ; metabolism ; Enzyme Precursors ; biosynthesis ; Genetic Vectors ; Larva ; metabolism ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; biosynthesis

Both phytase and endoglucanase are additives in feed for mono-gastric animal known for their effects. Recombinant vector pPICZalpha-EG was constructed and transformed to GS115-phyA, a Pichia pastoris strain that had integrated with phytase gene, generating GS115-phyA-EG. Both phytase and endoglucanase activities in the supernatant were determined after methanol induction of GS115-phyA-EG. Phytase and endoglucanase activity reached 39.4% and 56.2% activity compared to GS115-phyA and GS115-EG, respectively. Properties of the mixed enzyme suggest that the optimal temperature and pH value be 55 degrees C and 5.5 respectively. Both phytase and endoglucanase showed greater than 80% activity across temperature ranges 45 degrees C to 55 degrees C and pH ranges 4.5 to 5.5. Expressing more than one enzyme in one system could save time and money during induced expression, and the mixed enzyme might apply for treating forge before feeding with poultry.
6-Phytase ; biosynthesis ; genetics ; Cellulase ; biosynthesis ; genetics ; Genetic Vectors ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.
Animals ; Anti-Bacterial Agents ; biosynthesis ; Lactobacillus ; metabolism ; Lactoferrin ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Swine

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.
Escherichia coli ; Freeze Drying ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; Troponin C ; biosynthesis ; Troponin I ; biosynthesis

OBJECTIVETo express the L1 protein of human papillomavirus type 16 (HPV16) in insect cell suspension culture system.

METHODSOptimized the conditions of suspension culture, recombinant virus amplification and protein expression. Determined the virus tilter by plague analysis and detected the target protein by SDS-PAGE and Western blot; The formation of VLPs by HPV16 L1 protein was observed with TEM.

RESULTSThe Sf9 cells could grow better in suspension culture with seeding density of 5 x 10-5 cell/mL and the maximum expression quantity was obtained by infection of cells with rBacV/HPV16L1 (MOI =10) and harvesting after 72-84 h. HPV16L1 protein could assemble into VLPs in Sf9 cells observed with TEM.

CONCLUSIONThe conditions of cell culture, virus amplification and protein expression were optimized. HPV16 L1 protein could assemble into VLPs in Sf9 cells, which would provide a foundation for further study of the vaccine and diagnosis kits.

Animals ; Capsid Proteins ; biosynthesis ; Cell Proliferation ; Oncogene Proteins, Viral ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Spodoptera ; Suspensions ; Virion ; ultrastructure

The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.
Animals ; Bombyx ; genetics ; metabolism ; Chitin ; metabolism ; Insect Proteins ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; Silk ; biosynthesis

To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
Animals ; Cloning, Molecular ; Escherichia coli ; metabolism ; Fish Proteins ; biosynthesis ; Flounder ; Recombinant Fusion Proteins ; biosynthesis