Abstract:
:Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Jiao L,Zhou Q,Su Z,Xu L,Yan Ydoi
10.1016/j.pep.2018.02.005subject
Has Abstractpub_date
2018-07-01 00:00:00pages
1-12eissn
1046-5928issn
1096-0279pii
S1046-5928(17)30696-4journal_volume
147pub_type
杂志文章abstract::A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(92)90059-6
更新日期:1992-02-01 00:00:00
abstract::Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.08.008
更新日期:2011-12-01 00:00:00
abstract::The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide. The active site contains a critical metal ion such as manganese, iron, or copper. The copper-containing protein also has one zinc ion bound per subunit. The stand...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1241
更新日期:2000-06-01 00:00:00
abstract::Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli e...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.07.011
更新日期:2010-02-01 00:00:00
abstract::Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a clone...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.1015
更新日期:1999-03-01 00:00:00
abstract::Huperzine A (HupA) is a drug used for the treatment of Alzheimer's disease. However, the biosynthesis of this medicinally important compound is not well understood. The HupA biosynthetic pathway is thought to be initiated by the decarboxylation of lysine to form cadaverine, which is then converted to 5-aminopentanal b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.07.013
更新日期:2016-12-01 00:00:00
abstract::Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.05.003
更新日期:2004-09-01 00:00:00
abstract::Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in cli...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.08.005
更新日期:2007-12-01 00:00:00
abstract::The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Protea...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105725
更新日期:2020-12-01 00:00:00
abstract::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 methyltr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2018.05.012
更新日期:2018-10-01 00:00:00
abstract::The cDNA for the alpha i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques....
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1993.1010
更新日期:1993-02-01 00:00:00
abstract::Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-termi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.07.002
更新日期:2006-02-01 00:00:00
abstract::Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.09.005
更新日期:2017-01-01 00:00:00
abstract::The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastori...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.06.018
更新日期:2006-11-01 00:00:00
abstract::A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-s...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(90)90041-v
更新日期:1990-09-01 00:00:00
abstract::Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviou...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2021.105818
更新日期:2021-04-01 00:00:00
abstract::Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.07.002
更新日期:2015-10-01 00:00:00
abstract::Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a clea...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1553
更新日期:2002-02-01 00:00:00
abstract::Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The ta...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105746
更新日期:2021-01-01 00:00:00
abstract::Proteins are essential throughout the biological and biomedical sciences and the purification strategies of proteins of interest have advanced over centuries. Elastin-like polypeptides (ELPs) are compound polymers that have recently been highlighted for their sharp and reversible phase transition property when heated ...
journal_title:Protein expression and purification
pub_type: 杂志文章,评审
doi:10.1016/j.pep.2018.09.006
更新日期:2019-01-01 00:00:00
abstract::A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.1005
更新日期:1999-03-01 00:00:00
abstract::Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.04.001
更新日期:2005-08-01 00:00:00
abstract::Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is cr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.02.004
更新日期:2019-06-01 00:00:00
abstract::The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The bio...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.04.017
更新日期:2004-08-01 00:00:00
abstract::Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.07.016
更新日期:2013-10-01 00:00:00
abstract::A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purifi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.018
更新日期:2008-04-01 00:00:00
abstract::Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in o...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1054
更新日期:1999-08-01 00:00:00
abstract::The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Gl...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00015-9
更新日期:2003-06-01 00:00:00
abstract::High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZalphaA with the Saccharomyces cerevisiae alpha-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.06.021
更新日期:2009-11-01 00:00:00
abstract::Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab'...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(05)80037-3
更新日期:1992-10-01 00:00:00