Plasmid Construction

Categories:Protein Production
Time of issue:2020-08-14 00:00:00
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Description:

Information

Biortus routinely searches for the best expression conditions for a novel recombinant protein from the three major expression hosts (E. coli, Insect and mammalian), unless specified by clients or well-documented in literature. We offer a selection of plasmids and cells for achieving optimal expression. Here is an outline of parameters that we could try for optimal expression in E.coli:

1. Screening of Expression Tags and Plasmids

N-terminal Tags
6xHis-TEV
6xHis-GST-TEV
6xHis-MBP-TEV
6xHis-TrxB-TEV
6xHis-NusA-TEV
6xHis-Dsbc-TEV
6xHis-IF2-TEV
6xHis-FH8-TEV
6xHis-Ferritin-TEV

C-terminal Tag
-TEV-6xHis
-TEV-GFP-6xHis
-TEV-6xHis-10R
-TEV-GST-6His

Plasmids
pETDuet-1
pACYADuet-1
pCold TF
pGEX-4T
pET21a
pET28a
pET32a

2. Screening of Expression Cell Strains

BL21 (DE3)
Rosetta (DE3)
Origami 2 (DE3)
C41 (DE3)orC43 (DE3)
Arctic (DE3)
Tuner(DE3)0.1mM IPTG
BL21 (DE3) pLysS

most used
rare codon
-S-S-& rare codon
hydrophobic protein
TPN30 & TPN60 chaperone
lower expression for better folding
lower basal expression for toxic proteins

3. Screening with Chaperones

pG-KJE8,pGro7, pKJE7, pGTf2,pTf16

Dsba and/or Dsbc (expressed in periplasmic space with pET21a)