Bacterial Expression
vectors

Vector elements

promoters
solubility
fusions
affinity
tags
secretion
vectors

ATUM offers a range of bacterial expression vectors. Combinations of different promoters, selectable markers and copy numbers enables customers to choose a vector suited to their expression system.

Yield

Not sure how your protein will behave? Test your gene in ATUM's ready-to-use vectors with a variety of properties most likely to increase expression.

Speed

ATUM offers pre-selected Expression or Secretion panels, or you can create your own to quickly explore the properties you think are most important.

Precision

Combining promoters with different strength ribosome binding sites and origins of replication provides an excellent range of induced and uninduced expression levels.

Promoters

Overview - Promoters

Artboard 1 Medium High Ori Low Custom 1 Custom 2 Kan Zeo Gen Chlor Amp Marker ORF Promoter Rhamnose Arabinose PhoA T7 T5 RBS Medium Weak Strong Secretion ompC pelB mal Tag Fh8 GST MBP

ATUM offers rhamnose, IPTG (T5 and T7) and PhoA-based inducible bacterial expression vectors. Combining these with different strength ribosome binding sites and origins of replication provides an excellent range of induced and uninduced expression levels.

Promoter Properties

Promoter Inducer Strain
T5 IPTG-inducible, Repressible with 2% glucose Any E. coli
T7 IPTG-inducible, Repressible with 2% glucose BL21(DE3) or T7 Express
rham Rhamnose-inducible, Tight regulation and tunability, Repressible with 2% glucose Any E. coli
phoA Induction requires phosphate starvation, Repressible with 150µM phosphate. Excellent for low-cost autoinduction Any E. coli

Protein Expression from Different Promoters

Different promoters work better for different proteins. Expression of three proteins, phi29 (~61 kD), cutinase (~22 kD) and DasherGFP (~26 kD), under control of different promoters, T5 and T7 (IPTG-inducible), rham (rhamnose-inducible) and phoA (inducible by phosphate starvation) is shown. Expression values shown on the y-axis are measurements of expressed protein band densities from a SDS-PAGE gel using BSA as standard.

*DasherGFP measured fluorescence is lower with T7 and rham expressed GFP, indicating that a fraction of total protein is incompletely folded protein.

Independent Expression Using Two Orthogonal Promoters

Choice of promoters and inducible systems allow orthogonal expression of two proteins in a single strain by using a two vector system for induction control of each individual protein. Choose from vectors with promoters that are IPTG-inducible (T5 or T7), rhamnose-inducible, arabinose-inducible, or inducible phoA.

Orthogonal Expression of YFP and CFP in a Two Vector System:

  • Cloning of YFP and CFP into two pDAUGHTER vectors – rham_YFP and T5_CFP
  • No interference observed – expression is exclusive
  • Comparable expression with Rham and T5 promoters

Orthogonal Expression in a Single Strain

KringleYFP

Fluorescence of KringleYFP in pD8xx (pD884-12C, low copy) was measured at excitation/emission of 520/540 nm to exclude any background signal from CFP. This gives a better signal-to-noise ratio.

CindyLouCFP

Fluorescence of CindyLouCFP in pD444 was measured at excitation/emission of 395/500 nm.

Overview - Solubility Tags

Artboard 1 Medium High Ori Low Custom 1 Custom 2 Kan Zeo Gen Chlor Amp Marker ORF Promoter Rhamnose Arabinose PhoA T7 T5 RBS Medium Weak Strong Secretion ompC pelB mal Tag Fh8 GST MBP

Solubility-enhancing tags are generally large peptides or proteins that increase the expression and solubility of fusion proteins. No single fusion tag can increase the expression and solubility of all target proteins. Therefore, ATUM offers vectors with a variety of solubility tags. A comparison of fusion tags that help improve solubility of some difficult to express soluble proteins is shown below. Select a tag from the interactive graph or get the entire set to determine the best tag for your protein.

Available Tags

Tag Mol. Wt. Uses
GST 26 kD Solubility (N-term), Purification (Glutathione affinity or GST antibody), Detection (Western blot, quantitative assay based on enzymatic activity)
MBP 40 kDa Solubility (N-term), Purification (Amylose affinity with maltose elution)
PpiB 19 kDa Solubility (N-term)
Fh8 8 kDa Solubility (N-term), Purification (Hydrophobic column)
Custom Solubility, Purification or Detection

Effectiveness of Solubility Tags is Protein Dependent

Gene
Yield (nmole/µl)

Expression of total and soluble protein obtained using different tags. Three proteins encoded by genes A (~48 kD), B (~49 kD) and C (~59 kD), having shown poor solubility, were expressed in IPTG-inducible T5 promoter E. coli expression vectors (pD441-XXX) with various N-terminal solubility tags: MBP, GST, PpiB, and Fh8; 6XHis was included as a negative control. Cultures were grown at room temperature (RT) and 37°C. Overall, higher soluble protein expression was observed at RT (data shown). Percent solubility is shown along the Y-axis and the size of the circles corresponds to protein yield shown in nmole/µl. Legend shows approximate yield of protein based on circle size.

An integral part of tag choice is the method for removing the tag after purification. This step almost always involves using a protease to cleave a specific peptide bond between the tag and recombinant protein. ATUM offers the tobacco etch virus (TEV) protease with a 6XHis tag and the HRV3C protease with a GST tag to facilitate removal of the enzyme post-cleavage. Use the ATUM Expression Test to determine the best vector for your construct. ATUM will clone and test your construct in a full bacterial vector panel to quickly determine expression levels in E. coli.

Overview - Affinity Tags

Artboard 1 Medium High Ori Low Custom 1 Custom 2 Kan Zeo Gen Chlor Amp Marker ORF Promoter Rhamnose Arabinose PhoA T7 T5 RBS Medium Weak Strong Secretion ompC pelB mal Tag Fh8 GST MBP

Affinity tags are known epitopes that when fused to either the C- or N-terminus of a recombinant protein are highly efficient tools for purifying proteins from crude extracts. This enables highly selective capture and circumvents multistep purification processes. In addition, the tags can be used for detection and may also help enhance protein expression.

Available Tags

Tag Mol. Wt. Uses Protease Cleavage Site Vectors
GST 26 kDa Solubility (N-term), Purification (Glutathione affinity or GST antibody), Detection (Western blot, quantitative assay based on enzymatic activity) HRV3C (LEVLFQ/GP)
MBP 40 kDa Solubility (N-term), Purification (Amylose affinity with maltose elution) HRV3C (LEVLFQ/GP)
FLAG 1 kDa Detection (C- or N-term)
CometGFP 26 kDa Detection (C- or N-term)
6XHis 1 kDa Purification (Nickel-charged sepharose), Detection (C- or N-term) TEV* (ENLYFQ/G)
Custom Solubility, Purification, Detection

N-terminal fusions can also help with expression as they can iron out any secondary structure that interferes with expression.

An integral part of fusion tag choice is the method for removing the tag after purification. This step almost always involves using a protease to cleave a specific peptide bond between the tag and recombinant protein. ATUM offers the tobacco etch virus (TEV) protease with a 6XHis tag and the HRV3C protease with a GST tag to facilitate removal of the enzyme post-cleavage.

Overview - Secretion Signals

Artboard 1 Medium High Ori Low Custom 1 Custom 2 Kan Zeo Gen Chlor Amp Marker ORF Promoter Rhamnose Arabinose PhoA T7 T5 RBS Medium Weak Strong Secretion ompC pelB mal Tag Fh8 GST MBP

Why secreted expression?

Production of secreted recombinant proteins using E. coli rhaBAD promoters offers several advantages compared to cytosolic production, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. Since the use of a post-translational or co-translational export mechanism is protein specific and cannot be known a priori, vectors with different secretion signals allows for the selection of the translocation pathway best suited for a given recombinant protein. Note: ATUM recommends the use of vectors with rhamnose inducible promoters for secreted protein expression. There are many distinct advantages to the rhamnose secretion vectors:

  1. Expression of protein is tightly tunable with rhamnose concentration thus allowing you to modulate protein expression, so as to avoid overwhelming the secretion machinery.
  2. Rhamnose vectors are available with a wider selection of 18 secretion signals spanning the Sec, TAT, and SRP translocation pathways.

Vectors are also available with YebF to enable secretion into media

ATUM vectors with different secretion signals allow selection of the secretion signal best suited to each protein. Parallel processing of multiple pDAUGHTER secretion vectors allows a quick and convenient method for selecting the signal(s) which provides the highest levels of secreted expression. While different secretion signals have been used, individual levels of secretion may vary and depend upon the recombinant protein being expressed. There is no general rule to selecting a secretion signal to guarantee successful secretion for a given recombinant protein, as efficiency of secretion depends on host strain, signal sequence and type of protein.

Secretion Signals

Signal Sequences Amino Acid Sequence Length Promoters Vectors
dsbA MKKIWLALAGLVLAFSASA 19 aa Rham
EOX MFKFKKKFLVGLTAAFMSISMFSATASA 28 aa Rham
lamB MMITLRKLPLAVAVAAGVMSAQAMA 25 aa Rham
MglB MNKKVLTLSAVMASMLFGAAAHA 23 aa Rham
MmAp MKKNIIAGCLFSLFSLSALA 20 aa Rham
ompC MKVKVLSLLVPALLVAGAANA 21 aa Rham
ompT MRAKLLGIVLTTPIAISSFA 20 aa Rham
sufI MSLSRRQFIQASGIALCAGAVPLKASA 27 aa Rham
SfmC MMTKIKLLMLIIFYLIISASAHA 23 aa Rham
STII MKKNIAFLLASMFVFSIATNAYA 23 aa Rham
tolB MKQALRVAFGFLILWASVLHA 21 aa Rham
torA MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA 39 aa Rham
torT MRVLLFLLLSLFMLPAFS 18 aa Rham
gIII (minor capsid protein from fd phage) MKKLLFAIPLVVPFYSHS 18 aa T5, Rham
malE (maltose binding protein) MKIKTGARILALSALTTMMFSASALA 26 aa T5, Rham
ompA (outer membrane protein A) MKKTAIAIAVALAGFATVAQA 21 aa T5, Rham
pelB (pectate lyase B) MKYLLPTAAAGLLLLAAQPAMA 22 aa T5, Rham
phoA (alkaline phosphatase) MKQSTIALALLPLLFTPVTKA 21 aa T5, Rham
YebF* 117 aa Rham

*Proteins fused to YebF are secreted into the media.


Use the ATUM Expression Test to determine the best vector for your construct. ATUM will clone and test your construct in a full bacterial vector panel to quickly determine expression levels in E. coli.

Expression with Secretion Signals

Protein
Yield (µg/ml)

Different proteins prefer different secretion signals. Periplasmic expression of MBP, Cutinase, and Alkaline phosphatase from rhamnose vectors with different secretion signals. Total cellular protein levels are represented as circle diameter. Soluble protein in the periplasm is shown on the y-axis. Protein was quantified by densitometry of stained acrylamide gels.

Vector Cost


Vector Cost for Gene Synthesis Projects: Cloning of synthetic genes into the first vector is available free of charge. If the same gene is cloned into multiple vectors, additional fees will apply. Regular gene synthesis fees apply.

ATUM can even test your gene for high expression.

Expression Test is now available for bacterial vector panels with a volume discount. Cost for expression testing is $395.00 USD per gene per vector with an additional 10% off the total. Prices are in addition to regular gene synthesis fees.

Positive control fluorescent and chromogenic proteins in these vectors are available from ATUM’s catalog at half price if ordered with gene synthesis or Electra Vector orders.

Important Info: Mammalian Expression Vectors do NOT contain the multiple cloning sites. Restriction Sites: The pJ and pD vectors do not contain restriction sites for excision of your gene. Any restriction site desired can be added to your synthetic gene insert, and must be included in your gene design if you wish to remove your gene with restriction enzymes.

FH8 solubility tag is licensed from Hitag and is available for research use only. A separate license is required for any commercial use, including the development of commercial products or services. Information about commercial licenses may be obtained from Hitag Biotechnology. Lda, Biocant Park L4 N4, 3060-197, Cantanhede, Portugal.

References


View Publications using ATUM Bacterial Expression Vectors

Search the ATUM Literature Database,containing over 2,660 scientific publications using ATUM technology for references relevant to your research.

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