BL21-CodonPlus系列大腸桿菌菌株-感受態(tài)細(xì)胞-BioVector NTCC質(zhì)粒載體菌種細(xì)胞蛋白抗體基因保藏中心

INTRODUCTION

BL21-CodonPlus? competent cells* are derived from Stratagene’s highperformance

BL21-Gold competent cells.1 These cells enable efficient highlevel

expression of heterologous proteins in Escherichia coli.

Efficient production of heterologous proteins in E. coli is frequently limited

by the rarity of certain tRNAs that are abundant in the organisms from

which the heterologous proteins are derived.ll Forced high-level expression

of heterologous proteins can deplete the pool of rare tRNAs and stall

translation. BL21-CodonPlus strains are engineered to contain extra copies

of genes that encode the tRNAs that most frequently limit translation of

heterologous proteins in E. coli. Availability of tRNAs allows high-level

expression of many heterologous recombinant genes in BL21-CodonPlus

cells that are poorly expressed in conventional BL21 strains.

BL21-CodonPlus-RIL and BL21-CodonPlus(DE3)-RIL cells contain extra

copies of the argU, ileY, and leuW tRNA genes. These genes encode tRNAs

that recognize the arginine codons AGA and AGG, the isoleucine codon

AUA, and the leucine codon CUA, respectively (Table I). The CodonPlus-

RIL strains have available the tRNAs that most frequently restrict

translation of heterologous proteins from organisms that have AT-rich

genomes. BL21-CodonPlus-RP and BL21-CodonPlus(DE3)-RP cells

contain extra copies of the argU and proL genes. These genes encode

tRNAs that recognize the arginine codons AGA and AGG and the proline

codon CCC, respectively. The CodonPlus-RP strains have available the

tRNAs that most frequently restrict translation of heterologous proteins of

organisms that have GC-rich genomes. The BL21-CodonPlus (DE3)-RIPL

cells contain extra copies of the argU, ileY, and leuW as well as the proL

tRNA genes. This strain rescues expression of heterologous proteins from

organisms that have either AT- or GC-rich genomes.

Extra Copies of tRNA Genes in BL21-CodonPlus Strains

Host strain tRNA genes (codon recognition of gene product) Antibiotic

resistance

BL21-CodonPlus(DE3)-RIPL strain argU (AGA, AGG), ileY (AUA) , proL (CCC), leuW (CUA) Camr

Strep/Specr

BL21-CodonPlus-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) Camr

BL21-CodonPlus(DE3)-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) Camr

BL21-CodonPlus(DE3)-RIL-X strain argU (AGA, AGG), ileY (AUA), leuW (CUA) Camr

BL21-CodonPlus-RP strain argU (AGA, AGG), proL (CCC) Camr

BL21-CodonPlus(DE3)-RP strain argU (AGA, AGG), proL (CCC) Camr

BL21-CodonPlus(DE3)-RP-X strain argU (AGA, AGG), proL (CCC) Camr

The BL21-CodonPlus(DE3) strains (Table II) are ideal for performing

protein expression studies that utilize the T7 RNA polymerase promoter to

direct high-level expression. The BL21-CodonPlus-RIL and

BL21-CodonPlus-RP strains can be used for protein expression with vectors

driven by non-T7 promoters. The methionine auxotrophic variants

BL21-CodonPlus(DE3)-RIL-X and BL21-CodonPlus(DE3)-RP-X allow

efficient labeling of recombinant proteins with selenomethionine or

35S-methionine. Both strains contain a transposon in the metA gene that

renders the cells incapable of synthesizing methionine. Derived from E. coli

B, the BL21-CodonPlus expression strains naturally lack the Lon protease,

which can degrade recombinant proteins. In addition, these strains are

engineered to be deficient for a second protease, the OmpT protein.

BL21-CodonPlus competent cells also feature the Hte phenotype* present in

Stratagene's highest efficiency strain, XL10-Gold? ultracompetent cells.2

The presence of the Hte phenotype increases the transformation efficiency

of the cells. In addition, the gene that encodes endonuclease I (endA), an

enzyme that rapidly degrades plasmid DNA isolated by most miniprep

procedures, has been inactivated in these cells. These two features enable

direct cloning of many protein expression constructs.

Host Strains and Genotypes

Host straina Genotype

BL21-CodonPlus(DE3)-RIPL strain E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal λ(DE3) endA Hte [argU proL

Camr] [argU ileY leuW Strep/Specr]

BL21-CodonPlus-RIL strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal endA Hte [argU ileY leuW

Camr]

BL21-CodonPlus(DE3)-RIL strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal λ(DE3) endA Hte [argU ileY

leuW Camr]

BL21-CodonPlus(DE3)-RIL-X strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal λ(DE3) endA metA::Tn5(kanr)

Hte [argU ileY leuW Camr]

BL21-CodonPlus-RP strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal endA Hte [argU proL Camr]

BL21-CodonPlus(DE3)-RP strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal λ(DE3) endA Hte [argU proL

Camr]

BL21-CodonPlus(DE3)-RP-X strain3 E. coli B F– ompT hsdS(rB

– mB

–) dcm+ Tetr gal λ(DE3) endA metA::Tn5(kanr)

Hte [argU proL Camr]

a These strains, derivatives of E. coli B, are general protein expression strains that lack both the Lon protease and the

OmpT protease, which can degrade proteins during purification.4 Dcm methylase, naturally lacking in E. coli B, is

inserted into the genomes.

Table III

Expression strains Induction Advantages Disadvantages

BL21-CodonPlus-RIL

competent cells

BL21-CodonPlus-RP

competent cells

Infection with lambda

bacteriophage CE6

Tightest control of

uninduced expression

Induction is not as efficient as

in DE3 derivatives and

induction (infection) process

is more cumbersome

BL21-CodonPlus(DE3)-RIPL

competent cells

BL21-CodonPlus(DE3)-RIL

competent cells

BL21-CodonPlus(DE3)-RP

competent cells

Isopropyl-1-thio-β-Dgalactopyranoside

(IPTG) induction of T7

polymerase from

lacUV5 promoter

High-level expression Leaky expression of T7

polymerase can lead to

uninduced expression of

potentially toxic proteins

BL21-CodonPlus(DE3)-RIL-X

competent cells

BL21-CodonPlus(DE3)-RP-X

competent cells

Isopropyl-1-thio-β-Dgalactopyranoside

(IPTG) induction of T7

polymerase from

lacUV5 promoter

High-level expression

Efficient labeling of

recombinant proteins with

methionine derivatives

Leaky expression of T7

polymerase can lead to

uninduced expression of

potentially toxic proteins