Production of authentic geranylgeranylated KRAS4b using an engineered baculovirus system
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Lauren Procter, Carissa Grose, Dominic Esposito Protein prenylation is a vital eukaryotic post-translational modification which permits interaction of proteins with cellular membranes. Prenylated proteins are involved in a number of human diseases, and play a major role in cancers driven by the oncogene KRAS, which is normally farnesylated. In cases where the farnesylation machinery is inhibited, however, KRAS eludes inactivation by using an alternative prenylation pathway in which the protein is geranylgeranylated. In order ...
Source: Protein Expression and Purification - June 28, 2018 Category: Biochemistry Source Type: research

Expression and purification of the SNX1/SNX6 complex
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Xin Yong, Wenfeng Hu, Xue Zhou, Jing Wang, Ezra Burstein, Da Jia The sorting nexin (SNX) family proteins play an essential role in vesicular transport, cell signaling, and membrane remodeling. The SNX members SNX1/2 and SNX5/6 form dimers, and mediate endosome-to-trans Golgi network (TGN) transport through coordinating cargo selection and membrane remodeling. It is well-known how a SNX-BAR protein forms a homodimer; however, it is less clear how a heterodimer is formed. Here a detailed expression and purification protocol of ...
Source: Protein Expression and Purification - June 23, 2018 Category: Biochemistry Source Type: research

High-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Andrew G. Diamos, Hugh S. Mason Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the ...
Source: Protein Expression and Purification - June 23, 2018 Category: Biochemistry Source Type: research

Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Ulrike Blaschke, Beneditta Suwono, Sachli Zafari, Ingo Ebersberger, Evelyn Skiebe, Cy M. Jeffries, Dmitri I. Svergun, Gottfried Wilharm Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucida...
Source: Protein Expression and Purification - June 22, 2018 Category: Biochemistry Source Type: research

High-level extracellular protein expression in Bacillus subtilis by optimizing strong promoters based on the transcriptome of Bacillus subtilis and Bacillus megaterium
In this study, the transcriptomes of B. subtilis 168 and B. megaterium DSM319 cells grown in stationary phase were analyzed to expand the repertoire of highly-active promoters for high-level protein expression based on the transcriptomes of these Bacillus strains. 24 genes with the highest expression levels among 2048 highly expressed gene families were chosen to examine promoter activity. The activities of four promoters with the beta-galactosidase (bgaB) gene as a reporter were stronger than those of the well-characterized strong promoter P43. The expression level of recombinant Pro-transglutaminase (pro-MTG) from Stre...
Source: Protein Expression and Purification - June 21, 2018 Category: Biochemistry Source Type: research

Evaluation of in vitro refolding vs cold shock expression: Production of a low yielding single chain variable fragment
This study reports on: i) the improved expression of a previously low yielding TFI-scFv in the cytoplasm of E. coli BL21 (DE3) through modifications to the expression systems in conjunction with codon optimization ii) evaluation of two commercial methods of protein recovery: in vitro refolding and the utilization of cold shock expression systems in conjunction with E. coli SHuffle. Results showed that TFI-scFv could be expressed at higher levels in the cytoplasm of E. coli than previously achieved in the periplasm. Both the in vitro refolding and cold shock strategies were capable of producing functional TFI-scFv with vary...
Source: Protein Expression and Purification - June 20, 2018 Category: Biochemistry Source Type: research

Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Juanjuan Yang, Yindi Liu, Shuli Xu, Haiying Lin, Chun Meng, Donghai Lin The trans-translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans-translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis (MtbSmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works...
Source: Protein Expression and Purification - June 19, 2018 Category: Biochemistry Source Type: research

Expression and purification of pneumococcal surface protein a of clade 4 in Escherichia coli using hydroxylapatite and ion-exchange column chromatography
In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary st...
Source: Protein Expression and Purification - June 17, 2018 Category: Biochemistry Source Type: research

Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): B.B. Gengenbach, C.R. Müschen, J.F. Buyel Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in ...
Source: Protein Expression and Purification - June 14, 2018 Category: Biochemistry Source Type: research

Expression and purification of a rapidly degraded protein, TMEM8B-a, in mammalian cell line
In this study, we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression. 293FT cells were the best host cell line to express TMEM8B-a protein. Then, we treated the stable 293FT cell lines with various small-molecule inhibitors and demonstrated that treatment with MG-132 and bortezomib, which target the proteasome and disrupt its function, could prevent TMEM8B-a degradation and induce protein expression in 293FT cells. Finally, we utilized the combination o...
Source: Protein Expression and Purification - June 14, 2018 Category: Biochemistry Source Type: research

Cloning, expression, and functional analysis of lysine decarboxylase in mulberry (Morus alba L.)
In this study, the mulberry lysine decarboxylase gene (MaLDC), which is involved in the biosynthesis of DNJ alkaloids, was cloned, expressed, and functionally verified. MaLDC was induced and expressed in Escherichia coli BL21 (DE3). The recombinant soluble MaLDC protein had a relative molecular mass of 24.0 kDa. The protein was purified by Ni-NTA separation. The results showed that MaLDC protein could catalyze lysine decarboxylation to produce cadaverine. The Km and Vmax values were 19.2 μM and 3.31 μM/min, respectively. Quantitative real-time reverse transcription polymerase chain reaction revealed that MaLDC ...
Source: Protein Expression and Purification - June 13, 2018 Category: Biochemistry Source Type: research

Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 – A key enzyme for heparin biosynthesis
In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with ...
Source: Protein Expression and Purification - June 13, 2018 Category: Biochemistry Source Type: research

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Makoto Mizukami, Hiromasa Onishi, Hiroshi Hanagata, Akira Miyauchi, Yuji Ito, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao Tokunaga The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC ( Brevibacillus in vivo cloning) expression system. In the fed-batch high-densi...
Source: Protein Expression and Purification - June 9, 2018 Category: Biochemistry Source Type: research

Auto-induction for high level production of biologically active reteplase in Escherichia coli
Publication date: November 2018 Source:Protein Expression and Purification, Volume 151 Author(s): Mehrnoosh Fathi-Roudsari, Nader Maghsoudi, Amirhossein Maghsoudi, Sepideh Niazi, Morvarid Soleiman Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield g...
Source: Protein Expression and Purification - June 8, 2018 Category: Biochemistry Source Type: research

Expression of Anabaena sensory rhodopsin is influenced by different codons of seven residues at the N-terminal region
In this study, full-length ASR was used to test the influence of codon usage on expression E. coli. Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only...
Source: Protein Expression and Purification - June 5, 2018 Category: Biochemistry Source Type: research

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Miriam Grgic, Adele Williamson, Gro Elin Kjæreng Bjerga, Bjørn Altermark, Ingar Leiros Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replicatio...
Source: Protein Expression and Purification - May 31, 2018 Category: Biochemistry Source Type: research

Phosphorylated and non-phosphorylated HCK kinase domains produced by cell-free protein expression
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Kazushige Katsura, Yuri Tomabechi, Takayoshi Matsuda, Mayumi Yonemochi, Junko Mikuni, Noboru Ohsawa, Takaho Terada, Shigeyuki Yokoyama, Mutsuko Kukimoto-Niino, Chie Takemoto, Mikako Shirouzu Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant protein...
Source: Protein Expression and Purification - May 29, 2018 Category: Biochemistry Source Type: research

Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Yanan Guo, Miao Yu, Na Jing, Shoutao Zhang Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inse...
Source: Protein Expression and Purification - May 26, 2018 Category: Biochemistry Source Type: research

A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Richard R. Burgess Through years of research, teaching, troubleshooting, and editing, I have developed a solid working understanding of many aspects of protein purification and protein biochemistry. I have also found that many researchers do not understand the basics, let alone the pitfalls, of the techniques they use. In the case of size exclusion chromatography (SEC), I summarize below some of the rules of thumb, suggestions, limitations and troubleshooting I have found most useful in helping my students and colleagues carry...
Source: Protein Expression and Purification - May 26, 2018 Category: Biochemistry Source Type: research

Cloning, overexpression, and purification of a gene of unknown function of prophage loci from ‘Candidatus Liberibacter asiaticus,’ the destructive bacterial pathogen of huanglongbing disease in citrus plants
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Duangtip Sudhan, Thamrongjet Puttamuk, Supachai Vuttipongchaikij, Pitak Chuawong Citrus Huanglongbing (HLB) or citrus greening is one of the most destructive diseases affecting citrus industry worldwide. The causal agent in Asia is a phloem-limited, Gram-negative bacterium, 'Candidatus Liberibacter asiaticus' (CLas). Within the genome of CLas lies prophage regions, classified as Type-A, B, C, and D. In particular, Type-D has been indicated to correlate with the blotchy-mottle symptoms of citrus trees. Here we reported the clon...
Source: Protein Expression and Purification - May 25, 2018 Category: Biochemistry Source Type: research

High level expression and glycosylation of recombinant Mycobacterium tuberculosis Ala-Pro-rich antigen in Pichia pastoris
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Shengjun Wang, Yaoguang Wang, Peng George Wang, Min Chen, Yun Kong The Ala-Pro-rich Antigen (Apa) from Mycobacterium tuberculosis is a mannosylated protein with immunogenic and antigenic properties. The O-mannosylation is essential for its biological function in the process of colonization and invasion of host cells by M. tuberculosis. In this work, the gene encoding Apa was cloned from M. tuberculosis and expressed in Pichia pastoris GS115. In shake-flasks, the recombinant Apa was secreted into the culture media and purified ...
Source: Protein Expression and Purification - May 24, 2018 Category: Biochemistry Source Type: research

Expression of a functional intrabody against hepatitis C virus core protein in Escherichia coli and silkworm pupae
In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1–123 amino acid residues (C123), 1–152 amino acid residues (C152), 1–177 amino acid residues (C177) and 1–191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively,...
Source: Protein Expression and Purification - May 24, 2018 Category: Biochemistry Source Type: research

UDP-glucose pyrophosphorylase: Isolation, purification and characterization from developing thermotolerant wheat (Triticum aestivum) grains
Publication date: August 2018 Source:Protein Expression and Purification, Volume 148 Author(s): Deepika Balan, Jayanti Tokas, H.R. Singal UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) activity was determined in four different thermotolerant varieties of wheat viz. WH-1021, PBW-373, Raj-3765 and DBW-16. The specific activity of UGPase was found to be highest at 21 days after anthesis (DAA) in the variety WH-1021 which has been developed by Haryana Agricultural University, Hisar (Haryana, India). Hence, crude extract prepared from immature grains (21 days after anthesis) of WH-1021 was used for purification of UGPase u...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Antibody engineering to improve manufacturability
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Sujeewa D. Wijesuriya, Elizabeth Pongo, Milan Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, Shauna Farr-Jones, James D. Marks, Arnold H. Horwitz Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Cloning and characterization of ApCystatin, a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress
In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Mabfilin and Fabfilin - New antibody-scaffold fusion formats for multispecific targeting concepts
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Mathias Kahl, Florian Settele, Paul Knick, Ulrich Haupts, Eva Bosse-Doenecke Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules wit...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Rochelle Aw, Paul F. McKay, Robin J. Shattock, Karen M. Polizzi Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibod...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

A novel purification procedure for recombinant human serum albumin expressed in Pichia pastoris
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Shijie Li, Linlin Li, Zhuo Chen, Guangpu Xue, Longguang Jiang, Ke Zheng, Jincan Chen, Rui Li, Cai Yuan, Mingdong Huang Plasma-derived human serum albumin (pHSA) has important applications in many clinical indications, including blood loss, serious burn, or hemorrhagic shock. The limited supply and potential infectious pathogen contamination of pHSA have stimulated the development of recombinant human serum albumin (rHSA). However, rHSA often entraps endogenous or exogenous impurities, including color pigments and polysacchar...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Alexander J. Winton, Janae L. Baptiste, Mark A. Allen Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Effect of rare codons in C-terminal of green fluorescent protein on protein production in Escherichia coli
In this study, the structure-guided SCHEMA recombination method and site-specific mutagenesis were proposed to detect the contribution of the rare codons on the functional expression of eGFP. The 12 chimeric egfps were generated from egfp-codon and egfp-genscript by the software SCHEMA. The results indicated that it was the rare codons in the C-terminal coding region (residues from 147 to 239) of eGFP resulting in the higher expression levels in Escherichia coli. The single and multiple point mutations also indicated that the presence of rare codons in 3′ coding regions of egfp could enhance the functional expression...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ)
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Roopa Dharmatti, Hideyuki Miyatake, Chen Zhang, Xueli Ren, Akiko Yumoto, Daisuke Kiga, Masayuki Yamamura, Yoshihiro Ito Human folate receptors (hFRα and hFRβ) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requ...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Tomoka Kawase, Fumi Miura, Anusuya Debnath, Kinuyo Imakura, Shin-ichi Miyoshi Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein subst...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Secretory expression and scale-up production of recombinant human thyroid peroxidase via baculovirus/insect cell system in a wave-type bioreactor
In this study, to achieve improved secretory expression of hTPO via BEVS, a truncated recombinant hTPO protein (hTPOt) was engineered by replacing its original signal peptide (SP) in the N-terminal with five heterologous SPs. Our data showed that the SP from the peptidyl-glycine alpha-amidating monooxygenase (PAM), referred to as SPPAM, significantly promoted the secretion of SPPAM-fused hTPOt (p-hTPOt) in High Five cells. Subsequently, we established an optimized scale-up production procedure for p-hTPOt in a 5-L wave-type bioreactor. The secretory p-hTPOt was purified by immobilized metal-chelating affinity chromatograph...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

A novel l-leucine 5-hydroxylase from Nostoc piscinale unravels unexpected sulfoxidation activity toward l-methionine
Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Dengyue Sun, Dengke Gao, Panpan Xu, Qianqian Guo, Zhangliang Zhu, Xiaotao Cheng, Song Bai, Hui-Min Qin, Fuping Lu Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombin...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Improved high-yield expression, purification and refolding of recombinant mammalian prion proteins under aerosol-free elevated biological safety conditions
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Peter Rehbein, Harald Schwalbe Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prio...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Impairment of a membrane-targeting protein translated from a downstream gene of a “self-cleaving” T2A peptide conjunction
In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence. Two versions of the T2A-based vector were constructed by switching the DNA sequences encoding the proteins of interest, the N-myristoylated protein and the nuclear-homing protein, upstream and downstream of the 2A linker, respectively. Our results showed that similar levels of mRNA expression were found and 100% of cleavage efficiency of T2A was observed. Nevertheless, we also reported the cleared ev...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Zhenzhan Chang, Xiaoqiang Wang, Risheng Wei, Zhouying Liu, Hong Shan, Guizhen Fan, Hongli Hu Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Tian Zhao, Zhipeng Li, Zhouliang Guo, Ali Wang, Zhenxing Liu, Qing Zhao, Yuyin Li, Edward A. McKenzie, Aipo Diao Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant prot...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Satoru Shimada, Shintaro Maeda, Masahide Hikita, Kaoru Mieda-Higa, Shigefumi Uene, Yukiko Nariai, Kyoko Shinzawa-Itoh Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain th...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Purification, characterization, and stabilization of alcohol oxidase from Ogataea thermomethanolica
In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the V max, K m , and k cat of 0.24 nmol/min, 0.27 mM, and 3628.8 min−1, respectively against methanol. Addition of alginic acid (0.35%) showed...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Functional characterization of GH7 endo-1,4- β-glucanase from Aspergillus fumigatus and its potential industrial application
Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Aline Vianna Bernardi, Paula Fagundes de Gouvêa, Luis Eduardo Gerolamo, Deborah Kimie Yonamine, Laís de Lourdes de Lima Balico, Sergio Akira Uyemura, Taisa Magnani Dinamarco A gene encoding an endo-1,4-β-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residue...
Source: Protein Expression and Purification - May 20, 2018 Category: Biochemistry Source Type: research

Rapid purification of bacteriophage endolysin TSPphg and its exogenous treatment could act as an alternative bacterial cell disruption method
Publication date: August 2018 Source:Protein Expression and Purification, Volume 148 Author(s): Feng Wang, Xinyu Ji, Meishan Chen, Jun Guo, Xianyu Deng, Lianbing Lin Bacteriophage endolysins have long been demonstrated to be effective enzybiotics, and have the potential value in the areas of food, agricultural, and industrial science. Traditionally, extraction of recombinant proteins from bacterium E. coli is achieved by chemical, biological or mechanical disruption methods. Here, we present heat treatment, a simple and highly effective method that differs from the conventional ones, for disruption of E. coli cells to ext...
Source: Protein Expression and Purification - April 7, 2018 Category: Biochemistry Source Type: research

High yield production of human invariant chain CD74 constructs fused to solubility-enhancing peptides and characterization of their MIF-binding capacities
Publication date: August 2018 Source:Protein Expression and Purification, Volume 148 Author(s): Tjie Kok, Anna A. Wasiel, Frank J. Dekker, Gerrit J. Poelarends, Robbert H. Cool The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition o...
Source: Protein Expression and Purification - April 6, 2018 Category: Biochemistry Source Type: research

Facilitation of yeast-lethal membrane protein production by detoxifying with GFP tagging
Publication date: August 2018 Source:Protein Expression and Purification, Volume 148 Author(s): Hiroyuki Oshikane, Masahiko Watabe, Toshio Nakaki Recombinant techniques for target protein production have been rapidly established and widely utilised in today's biological research. Nevertheless, methods for membrane protein production have yet to be developed, since membrane proteins generally tend to be expressed at low levels, easily aggregated, and/or even toxic to their host cells. Here we report that a GFP-tagging technique can be applied for the stable production of membrane proteins that are toxic to their host cells...
Source: Protein Expression and Purification - April 6, 2018 Category: Biochemistry Source Type: research

A novel strategy to produce high level and high purity of bioactive IL15 fusion proteins from mammalian cells
In this study, we report that Lysine 86 in IL15 is responsible for the instability in mammalian cells when its C-terminus is fused to the albumin binding scFv (IL15-A10m3). We demonstrate that K86A or K86R mutants increased the expression of the fusion protein from HEK293 cells. When the wild type IL15 is used for the fusion, no recombinant IL15 fusion was detected in the culture media. Additionally, we determined that the residue 112 in IL15 is critical for the bioactivity of IL15-A10m3. Examination of single and double mutants provides a better understanding of how IL15 engages with its receptor complex to achieve full...
Source: Protein Expression and Purification - April 6, 2018 Category: Biochemistry Source Type: research

Improved inducible expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis by enhancer regulation
In this study, we investigated the effect of the enhancers DegQ, DegU, and DegS on pullulanase expression in a recombinant B. subtilis inducible system. The genes degQ, degU, and degS were introduced to the recombinant plasmid pMA0911-PsacB-pul harboring the promoter PsacB, signal peptide LipA, and gene encoding pullulanase. The regulatory effects of the enhancers involved in recombinant plasmids on pullulanase expression level were evaluated in B. subtilis WB600 and WB800, respectively. The positive regulation of DegQ toward pullulanase expression was detected from B. subtilis WB800, leading to a 60% increase in enzyme ac...
Source: Protein Expression and Purification - April 6, 2018 Category: Biochemistry Source Type: research

Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica
In this study, the full-length gene of G6PD from Y. lipolytica (YlG6PD) was cloned without intron and heterogeneously expressed in E. coli. Then, YlG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YlG6PD was completely dependent on NADP+ and its apparent K m for NADP+ was 33.3 μM. The optimal pH was 8.5 and the maximum activity was around 47.5 °C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 °C for 20 min YlG6PD activity was competitively inhibited by NADPH with a K i value of 56.04 μM. Most of the metal...
Source: Protein Expression and Purification - April 6, 2018 Category: Biochemistry Source Type: research

Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli
We report a method of purifying activated human MMP1 in E. coli without using urea or 4-Aminophenylmercuric acetate (APMA). Instead, a non-ionic detergent, Triton X-100, was used in the lysis buffer to solubilize MMP1 followed by the protease activities of both trypsin and MMP1 to digest E. coli proteins and activate pro-MMP1. Identity of activated MMP1 was confirmed by Western blot using anti-human MMP1 antibodies, whereas the mass was determined to be 43 kD using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS). Collagen and gelatin degradation by purified MMP1 were confirmed by...
Source: Protein Expression and Purification - April 5, 2018 Category: Biochemistry Source Type: research

High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris
Publication date: August 2018 Source:Protein Expression and Purification, Volume 148 Author(s): Ye Ke, XiaoMei Yuan, JiaSheng Li, Wei Zhou, XiaoHui Huang, Tao Wang An alkaline protease (Ap) was cloned from Aspergillus sojae GIM3.33 via RT-PCR technique. A truncated Ap without the signal peptide was successfully expressed in the Pichia pastoris KM71 strain. The following describes the optimal process conditions for the recombinant engineering of a strain expressing a recombinant Ap (rAp) in a triangular flask: inoculum concentration OD600 value 20.0 in 40 mL working volume (in 500 mL flasks), methanol addition (1.0%; v...
Source: Protein Expression and Purification - March 31, 2018 Category: Biochemistry Source Type: research

Rapid NMR-scale purification of 15N,13C isotope-labeled recombinant human STIM1 coiled coil fragments
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER – plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapi...
Source: Protein Expression and Purification - March 19, 2018 Category: Biochemistry Source Type: research