Using overlap‐extension PCR technique to fusing genes for constructing recombinant plasmids

Lu, Yuanyuan; Xiao, Shuqin; Yuan, Mingyue; Gao, Yibo; Sun, Jiaying; Xue, Chunsheng

doi:10.1002/jobm.201700455

Cited by 12 publications

(9 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To generate full-length antibodies, the V H and V L of humanized scFvs were fused with the constant region of heavy chain (HC) and kappa light chain of human IgG1 (LC, Accession number: ABU90709.2) by overlap-extension PCR (OE-PCR) [ 43 ], respectively. Primers used for OE-PCR were listed in Table S5 .…”

Section: Methodsmentioning

confidence: 99%

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Bai

Mei

et al. 2021

Biomedicines

View full text Add to dashboard Cite

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (−54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

show abstract

Section: Methodsmentioning

confidence: 99%

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Bai

Mei

et al. 2021

Biomedicines

View full text Add to dashboard Cite

show abstract

“…The C . lunata CX‐3 ( MAT1‐2 ) (INSDC: JFHG00000000) (Gao et al ., 2014) and ZD958 strains ( MAT1‐1 ) (a laboratory strain) (Lu et al ., 2018a) were used as WT for transformation and crosses in this study. Single and double ClFTR1 , ClNPS6 deletion mutant strains were generated using the CX‐3 strain.…”

Section: Methodsmentioning

confidence: 99%

“…To generate complementary constructs of ClFTR1‐ C and ClNPS6‐ C, the genes sequence containing the ClFTR1 and ClNPS6 gene were amplified (ClFTR1‐F2/ClFTR1‐R2, ClFTR1‐F4/ClFTR1‐R4, ClNPS6‐F2/ClNPS6‐R2 and ClNPS6‐F4/ClNPS6‐R4) using overlapping extension PCR (Table S1) (Lu et al ., 2018a). The resulting PCR products were ligated into the neomycin phosphotransferase cassette released from pII99.…”

Section: Methodsmentioning

confidence: 99%

The key iron assimilation genesClFTR1,ClNPS6were crucial for virulence ofCurvularia lunatavia initiating its appressorium formation and virulence factors

Sun

Gao

et al. 2020

Environmental Microbiology

Self Cite

View full text Add to dashboard Cite

Summary Iron is virtually an essential nutrient for all organisms, to understand how iron contributes to virulence of plant pathogenic fungi, we identified ClFTR1 and ClNPS6 in maize pathogen Curvularia lunata (Cochliobolus lunatus) in this study. Disruption of ClNPS6 significantly impaired siderophore biosynthesis. ClFTR1 and ClNPS6 did mediate oxidative stress but had no significant impact on vegetative growth, conidiation, cell wall integrity and sexual reproduction. Conidial germination delayed and appressoria formation reduced in ΔClftr1 comparing with wild type (WT) CX‐3. Genes responsible for conidial germination, appressoria formation, non‐host selective toxin biosynthesis and cell wall degrading enzymes were also downregulated in the transcriptome of ΔClftr1 and ΔClnps6 compared with WT. The conidial development, toxin biosynthesis and polygalacturonase activity were impaired in the mutant strains with ClFTR1 and ClNPS6 deletion during their infection to maize. ClFTR1 and ClNPS6 were upregulated expression at 12–24 and 48–120 hpi in WT respectively. ClFTR1 positively regulated conidial germination, appressoria formation in the biotrophy‐specific phase. ClNPS6 positively regulates non‐host selective toxin biosynthesis and cell wall degrading enzyme activity in the necrotrophy‐specific phase. Our results indicated that ClFTR1 and ClNPS6 were key genes of pathogen known to conidia development and virulence factors.

show abstract

“…Primers ( Table 2) were designed based on the framework region sequence from the parental AP2. Subsequently, overlapextension PCR (OE-PCR) [34] was performed to amplify and assemble the cross-cloned gene fragments containing two or more optimized CDRs by using PrimeSTAR Ò HS DNA Polymerase (TaKaRa, Dalian, China). These amplified scFv fragments were digested with restriction enzyme Nco Ⅰ and Hind III (TaKaRa, Dalian, China), and cloned into the prokaryotic expression vector pET-27b (Novagen, Madison, WI, USA).…”

Section: Combination Of Improved Cdrs From Various Scfv Mutants (Crosscloning)mentioning

confidence: 99%

Development of a novel, fully human, anti-PCSK9 antibody with potent hypolipidemic activity by utilizing phage display-based strategy

Lei

Chen

et al. 2021

EBioMedicine

View full text Add to dashboard Cite

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favourable druggability by utilizing phage display-based strategy. Methods: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a fulllength Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. Findings: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a K D as low as 1.42 nM, and a dramatically slow dissociation rate (k off , 4.68 £ 10 À6 s À1 ), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC 50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P = 0.658, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively. Interpretation: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.

show abstract

Using overlap‐extension PCR technique to fusing genes for constructing recombinant plasmids

Cited by 12 publications

References 6 publications

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

The key iron assimilation genesClFTR1,ClNPS6were crucial for virulence ofCurvularia lunatavia initiating its appressorium formation and virulence factors

Development of a novel, fully human, anti-PCSK9 antibody with potent hypolipidemic activity by utilizing phage display-based strategy

Contact Info

Product

Resources

About