Amended on 29 December 2018

Conclusion: according to my protein research as below, eucaryotic gene should be expressed by using eucaryotic cells.

I have found that the expression of genes in adult humans is surely under the control of various virus (my unpublished observation; but please see my site "Questions" in this RG).

Therefore, the gene expression and/or protein expression in procaryote bacteria seems to be under the control of bacterial virus/bacteriophage/bacterial plasmid.

I have recently found that Lactobacillus casei (Shirota) (purified from the beverage Yakult (Yakult Honsha Co. Tokyo, Japan)) has c.a. 12% of bacteriophage proteins (such as DNA-directed RNA polymerase of Bacteriophage SP6) among total bacterial-cell proteins (my unpublished observation using PDMD (Protein-Direct-Microsequencing-Deciphering) method; please see the file, HepG2 Fucoidan).

I have compared molecular weight of proteins between bacteria and humans (without considering glycochains).

Bacteria of Lactobacillus casei (Shirota) has Median 59,000 (Range; 24,000 - 230,000). Mean/Average = 74,400.

Wall-associated protein (Mr 230,000) and Proline dehydrogenase (Mr 149,000) are large Mr proteins.

Fetal hepatocyte Hc (American Caucasoid male and female) has Median 66,000 (Range; 14,000 - 666,000). Mean/Average = 94,300. Thus, Fetal human cells surely resemble to procaryote. It is noteworthy that fetal Hc has not expressed Connectin at all.

Desmoyokin/Neuroblast differentiation-associated protein AHNAK (Mr 666,000), Cadherin EGF LAG seven-pass G-type receptor 3/Flamingo homolog 1/EGF-like protein 1/Cadherin family member 11 (Mr 374,000), Chromatin-helicase-DNA-binding protein 6 (Mr 307,000), and SEN1 homolog/Probable helicase senataxin (Mr 303,000) are large Mr proteins.

Adult tissues have Connectin.

Connectin/titin has Mr 3,880,000. However, this is not so large molecule in humans; i.e., Mr of Glycoprotein Mucin-16 is 9,000,000 and Mr of Glycosaminoglycan Hyaluronic acid is 7, 200,000. These large Mr molecules are surely biosynthesized within human cells.

Healed hepatocyte HepG2 (15y, Caucasoid male; this patient has sadly died; cultured with fucoidan for 3 days) has Median 84,500 (Range; 9,700 - 3,880,000). Mean/Average = 127,100. The molecular weight pattern is very similar to Hepatoma HepG2. Thus, this result indicates that Fucoidan effect is not due to Gene expression at all (please see file again; HepG2 Fucoidan).

Mucin-16/Ovarian cancer related tumor marker CA125 (Mr 1,640,000), Baculoviral IAP repeat-containing protein 6/Ubiquitin-conjugating BIR domain enzyme apollon (Mr 549,000), Apolipoprotein B-100/Apo B-100 (Mr 516,000), Ankyrin-3/ANK-3 (Mr 495,000), Ankyrin-2/Brain ankyrin (Mr 447,000), A-kinase anchor protein 9/Centrosome- and Golgi-localized PKN-associated protein (Mr 442,000), Laminin subunit alpha-2/Merosin heavy chain (Mr 353,000), G-protein coupled receptor 112/Adhesion G-protein coupled receptor G4 (Mr 348,000), Cadherin EGF LAG seven-pass G-type receptor 1/Flamingo homolog 2 (Mr 340,000), A-kinase anchor protein 13/Lymphoid blast crisis oncogener protein (Mr 318,000), and Probable helicase senataxin (Mr 303,000) are large Mr proteins.

Humans (normal liver with pseudo liver cancer) has Median 68,000 (Range; 16,700 - 3,880,000). Mean/Average = 132,000.

Ryanodine receptor, skeletal muscle/RyR1 (Mr 570,000), Sacsin/DnaJ homolog subfamily C member 29 (Mr 520,000), Apolipoprotein B-100/Apo B-100 (Mr 516,000), and Dystrophin (Mr 416,000) are large Mr proteins.

Humans (LC-tissue named as No.6; this patient has survived) has Median 64,500 (Range; 11,900 - 3,880,000). Mean/Average = 124,000.

Ryanodine receptor, skeletal muscle/RyR1 (Mr 570,000), Fibrillin-1 (Mr 324,000) and Centromeric protein E/CENP-E/Kinesin-7 (Mr 305,000) are large Mr proteins.

Humans (HCC-tissue named as No.6; this patient has survived) has Median 67,000 (Range; 6,600 - 3,880,000). Mean/Average = 124,800. Therefore, this patient's liver has not been the true cancer or true HCC. This patient's liver has no HIV-1, but has SIV plus HCV.

Mucin 2/Intestinal mucin-2 (Mr 585,000), Apolipoprotein B-100/Apo B100 (Mr 516,000), and Centromeric protein E/CENP-E (Mr 305,000) are large Mr proteins.

HCC-tissue (with PBC; this patient has sadly died) has Median 69,000 (Range; 15,400 - 3,880,000). Mean/Average = 140,500. This liver has HIV-1 and HCV.

Laminin subunit alpha-2/Merosin heavy chain (Mr 353,000) and Adenomatous polyposis coli protein/APC protein/Deleted in polyposis 2.5 (Mr 321,000) are large Mr proteins.

Humans (LC-tissue with leprosy) has Median 66,500 (Range; 13,000 - 3,880,000). Mean/Average = 124,950. Apolipoprotein B-100/Apo B-100 (Mr 516,000), Zinc finger protein HRX/Histone-lysine N-methyltransferase 2A (Mr 450,000), Laminin alpha-1 chain/Laminin A chain (Mr 350,000), Adenomatous polyposis coli protein/APC protein/Deleted in polyposis 2.5 (Mr 320,000), Neurofibromin/Neurofibromatosis-related protein NF-1 (Mr 320,000) are large Mr proteins.

It is found that the Adenomatous polyposis coli protein/APC protein/Deleted in polyposis 2.5 is expressed only two females of HCC-tissue with PBC at 6.4 μg/mg tissue protein and LC-tissue with leprosy at 26.0 μg/mg tissue protein, respectively.

LC-tissue with leprosy has up regulating virus of Genome polyprotein (HAV) at 14.4 μg/mg tissue protein, and down regulating picornavirus is not present. HCC-tissue with PBC has up regulating virus of Genome polyprotein (HAV) at 24.0 μg/mg tissue protein, and has down regulating picornavirus of Genome polyprotein (Foot-and-mouth disease virus/FMDV) at 15.1 μg/mg tissue protein. This result surely indicates that protein expression in adult human cells is under the control of vira.

Hepatoma HepG2 (15y, Caucasoid male; this patient has sadly died) has Median 79,000 (Range; 12,400 - 3,880,000). Mean/Average = 125,000. This liver has HIV-1 and HCV. This result shows that Median is better indicator than Mean/Average.

Usher syndrome type-2A protein/Usherin (Mr 588,000), Epiplakin/450 kDa epidermal antigen (Mr 575,000), Ryanodine receptor 1/RyR1(Mr 570,000), Ankyrin-2/Brain ankyrin (Mr 447,000), Protocadherin-16 (Mr 373,000), Laminin alpha-2 chain/Merosin heavy chain (Mr 353,000), Hornerin (Mr 322,000), PDZ domain-containing protein 2/Activated in prostate cancer protein (Mr 321,000), and Pinin (Mr 321,000) are large Mr proteins.

It is noteworthy that cancer marker proteins are not present in Humans; i.e., Immuno therapy to human cancer can not be possible. However, it is also noteworthy that disappearance of Apo B-100 is a marker of true liver cancer to leading patients to death (HepG2 and HCC tissue with PBC) (please see file again; HepG2 Fucoidan). This disappearance of Apo B-100 in HCC tissues seems to be occurred via the gene repression by Pegivirus of GB virusC/GBV-C (HepG2; American Caucasoid; male) and Pestivirus of Hog cholera virus/CSFV (HCC tissue with PBC; Japanese; female), respectively (please see file; The Fascio effect). This sugests that cholesterol homeostasis is essential to survive. Therefore, I have very surprized that CSFV and GBV-C are the real murder virus to cancer patients.

Precise Apo B-100 expression has been up regulated by Flavivirus, Hepacivirus, Alphavirus/Togavirus, and HHV-1, and down regulated by Pestivirus and Pegivirus. Flavivirus are Louping ill virus/LIV, Dengue virus/DENV, and Japanese enkephalitis virus/JEV. Alphavirus/Togavirus are O'nyong Nyong virus/ONNV and Venezuelan equine enkephalitis virus/VEEV.

HIV-1, HCV, and GBV-C have been dramatically disappeared by edible Japanese Fucoidan (at 0.102 mg/mL for 3 days) (please see again the file; HepG2 Fucoidan). Therefore, fucoidan can repress the enlargement of tumor, and also can be the lifesaving drug.

Our result suggests that the gene expression in humans is performed by different mechanism than notorious "Operon Hypothesis" (presented by French Geneticist Francois Jacob).

Markers of cancer are changes in glycochain structure (due to attachment of L-Fucose) and reduction of membrane structures. These changes are induced by virus such as HCV and HIV-1. Thus, edible Japanese Fucoidan is only the safe anticancer drug or foodstuff (please see file; Rat DEN Np-Fuco).

The Median values are compared between cancer and non-cancer by Mann-Whitney's U test. The result has indicated that the cancer organ expresses higher molecular weight proteins (n1=5, n2=2; p < 0.05, by one-shoulder estimation). It is noteworthy that Fucoidan does not influence to the gene expression as assessed by their Median molecular weights of cellular proteins.

It is intersting that true cancer cells seem to have higher molecular weight proteins (using Median vaues) as compared to non-cancer cells.

Further, Connectin/titin (molecular weight 3,880,000) seems not possible to be synthesized by bacterial protein synthetic machinery.

By the way, I have found that only Casein (phospho protein), beta-Lactoglobulin (except alpha-S2 Casein and beta-Lactoglobulin, which are present in cattle, but not present in humans at all), Albumin, Connectin/titin (phospho protein), Haemoglobin, and Lysozyme (Tubulin and Actin are absent in humans) are not exceptionally glycoproteins. Therefore, I wonder why many of human glycoproteins are able to be produced in procaryotic cells?

Further, human kidney biotinidase has been purified from a healthy Italian male's urine, and protein sequence has been determined as AVPPQVGNGQEGQTNYYYVAAYIKPLKPLQLLGSEDT- (please see file; JMBT alopecia). On the other hand, fetal/inflammatory biotinidase of USA has been found to be AHTGEESVADHHEAE- (Cole H, Reynolds TR, Lockyer JM, Buck GA, Denson T, et al. (1994) Human serum biotinidase. cDNA cloning, sequence, and characterization. J Biol Chem 269: 6566-6570.). This suggests that organ differences in biotinidase are present in humans.

Human milk biotinidase has been purified from 2 Japanese women's breast milks, and sequence has been determined as [F, V]-PSYVA-[V, N]-TK-[V, D]-VPPYGYYVAAVYEP-[N, Q]-[L, H]-IQYMG-[C, P]--GYSXAIN-. This result suggests that the gene differences among individuals are also present in humans.

The racial differences also may be present in human genes and/or proteins, although we have not fully studied yet (please see file; Race BIN urine).

Furthermore, I have considering that eucaryotic proteins/glycoproteins are differently biosynthesized from procaryotic microbes such as Escherichia coli. This idea has been firstly presented by famous American Biochemist Dr. Hans Neurath. He has said that human proteins are synthesized via reversed reaction of proteases. This idea can uniquely explain why prion glycoprotein (containing two N-type glyco-chains) can multiply without help of DNA.

Enzyme reaction seems to be unexpectedly very specific. Human milk biotinidase is made at mammary gland, and has O-type glycochains.

Human kidney biotinidase has also O-type glycochains (please see file; Dr. Terentyeva Urine BIN). Human serum biotinidase has N-type glycochains (please see file; Compa mBIN). Therefore, glycochain attachment by biotinidase synthesizing enzyme seems to be very strictly performed without help of DNA information.

It is noteworthy that eucaryote has famous Ubiquitin－Proteasome system, which is absent in procaryote. The Ubiquitin－Proteasome system specifically degrade the old proteins. Therefore, protein synthesis and degradation system is clearly different between procaryote and eucaryote.

Therefore, I have determined and compared protein synthetic system and protein degradation system in the cultured cells.

Fetal hepatocyte Hc has protein synthetic system and protein degradation system at 44.40 and 4.8 μg/mg of cell protein, respectively.

Hepatoma HepG2 (cultured without fucoidan) has protein synthetic system and protein degradation system at 52.42 and 10.83 μg/mg of cell protein, respectively.

Healed normal hepatocyte HepG2 (cultured with fucoidan for 3 days) has protein synthetic system and protein degradation system at 40.47 and 38.74 μg/mg of cell protein, respectively.

It is found that fetal developing cells and cancer cells have low amount of protein degradation system.

Following is the precise results; i.e.,

Fetal hepatocyte Hc has protein degradation enzymes of only one enzyme of Erasin/UBX domain-containing protein 4 at 4.8 μg/mg of cell protein. This cell has protein synthetic enzymes of Vitamin K-dependent protein C/Protein C at 5.0, Kidney-type biotinidase at 1.9, Serum biotinidase at 1.9, Milk-type biotinidase at 1.7, Fetal-Inflammatory-type biotinidase at 0.23, LON peptidase N-terminal domain and RING finger protein 1/RING finger protein 191 at 7.2, Glutathione S-transferase A4/GST class-alpha member 4 at 13.6, and Chymase/Mast cell protease I at 12.9 μg/mg of cell protein, respectively.　　　

Hepatoma HepG2 (cultured without fucoidan) has protein degradation enzymes of E3 ubiquitin-protein ligase TRIM33/Transcription intermediary factor 1-gamma/Tripartite motif-containing protein 28 at 3.9, E3 ubiquitin-protein ligase RNF123/RING finger protein 123 at 3.4, Tankyrase-1 at 1.9, U4/U6,U5 tri-snRNP-associated protein 2 at 0.4, WW domain-containing oxidoreductase/Fragile site FRA16D oxidoreductase at 0.47, and Zinc finger Ran-binding domain-containing protein 1/ Ubiquitin thioesterase ZRANB1 at 0.76 μg/mg of cell protein, respectively. This cell has protein synthetic enzymes of ADAMTS-19/A disintegrin and metalloproteinase with thrombospondin motifs 19 at 5.6, ADAMTS-13/A disintegrin and metalloproteinase with thrombospondin motifs 13/von Willebrand factor-cleaving protease at 9.2, Brain-specific serine protease 4 at 0.9, Endoplasmic reticulum aminopeptidase 1/Aminopeptidase PILS at 0.53, Glutathione synthetase at 0.055, Metallocarboxypeptidase D/Carboxypeptidase D at 2.4, Nardilysin/N-arginine dibasic convertase at 14.0, Serum biotinidase at 18.2, Syntaxin-12 at 0.23, and Transmembrane protease serine 5/Spinesin/TMPRSS5 at 1.3 μg/mg of cell protein, respectively.　

Healed normal hepatocyte HepG2 (cultured with fucoidan for 3 days) has protein degradation enzymes of 26S Protease regulatory subunit 6B/Tat-binding protein 7/Proteasome 26S subunit ATPase 4 at 1.4, Arginyl-tRNA--protein transferase 1/Arginyltransferase 1 at 1.49, Baculoviral IAP repeat-containing protein 6/Ubiquitin-conjugating BIR domain enzyme apollon at 3.9, BRG1-associated factor 170/SWI/SNF complex subunit SMARCC2 at 4.7, Calcium/calmodulin-dependent protein kinase type 1/Calmodulin-like skin protein at 0.53, Deubiquitinating enzyme 19 at 10.7, Deubiquitinating enzyme 29 at 1.5, G2/mitotic-specific cyclin-B3 at 5.9, HIV-1 Nef-interacting protein/TNFAIP3-interacting protein 1/Virion-associated nuclear shuttling protein at 4.1, Leukemia-associated protein 5/E3 ubiquitin-protein ligase TRIM13/RING finger protein 77 at 0.51, Proteasome subunit beta type-1/Macropain subunit C5 at 0.26, Nesca/RUN and SH3 domain-containing protein 1at 2.7, and TNF receptor-associated factor 2/Tumor necrosis factor type 1 receptor-associated protein 2/26S proteasome non-ATPase regulatory subunit 2;/Protein 55.11 at 1.05 μg/mg of cell protein, respectively. This cell has protein synthetic enzymes of Kidney-type biotinidase at 4.8, Serum biotinidase at 9.8, Serine protease TADG-15/Suppressor of tumorigenicity 14 protein at 1.2, Neuroligin-1 at 5.2, Neprilysin at 1.2, Matrix metalloproteinase-19 at 1.3, Gelatinase B/Matrix metalloproteinase-9 at 2.8, Endothelin-converting enzyme-like 1 at 8.5, Dipeptidyl-peptidase 1/Cathepsin C at 0.09, Cathepsin F at 0.08, ADAMTS-like protein 2 at 3.6, and ADAMTS-4/Aggrecanase-1 at 1.9 μg/mg of cell protein, respectively.

Furthermore, I have determined and compared protein synthetic system and protein degradation system in the diseased human livers.

Normal liver (diagnosed as Pseudo liver cancer) has protein synthetic system and protein degradation system at 51.4 and 30.8 μg/mg of tissue protein, respectively. This liver seems to be normal. Therefore, this estimation has found to be the good indicator of cancer.

LC tissue (with leprosy) has protein synthetic system and protein degradation system at 84.78 and 2.1 μg/mg of tissue protein, respectively.

HCC tissue (with PBC) has protein synthetic system and protein degradation system at 46.6 and 3.0 μg/mg of tissue protein, respectively.

LC tissue (named as No.6) has protein synthetic system and protein degradation system at 49.6 and 1.35 μg/mg of tissue protein, respectively.

HCC tissue (No.6) has protein synthetic system and protein degradation system at 65.49 and 7.46 μg/mg of tissue protein, respectively.

Therefore, LC tissue is considered to be already state of cancer (HCC) in protein metabolism.

Following is the precise results about liver tissues; i.e.,

Normal liver (diagnosed as Pseudo liver cancer) has protein degradation enzymes of E3 Ubiquitin protein ligase Praja-1/RING finger protein 70/PJA1 at 21.1, TAT-binding protein-1/TBP-1/26S protease regulatory subunit 6A at 2.3, and Ubiquitin carboxyl-terminal hydrolase 36/Ubiquitin thioesterase 36 at 7.4 μg/mg of tissue protein, respectively.

This tissue has protein synthetic enzymes of Acylamino-acid-releasing enzyme/APH at 2.0, Beta-Secretase 1 at 3.9, Complement factor I at 2.0, Mitochondrial intermediate peptidase/MIP at 2.7, Serum biotinidase at 22.7, Kidney-type biotinidase at 16.0, and Vitamin K-dependent protein S at 2.1 μg/mg of tissue protein, respectively.

LC tissue (with leprosy) has protein degradation enzymes of Protein MSS1/26S protease regulatory subunit 7 at 2.1 μg/mg of tissue protein.

This tissue has protein synthetic enzymes of Aminopeptidase N/Membrane alanyl aminopeptidase/GP150;/CD13 at 4.9, Bone morphogenetic protein 1/BMP-1 at 5.0, Carboxypeptidase B/Pancreas-specific protein at 0.66, Cathepsin B at 3.2, Coagulation factor V/Activated protein C cofactor at 5.5, Elastase 3A, pancreatic at 0.66, Elastase IIIB/Chymotrypsin-like elastase family member 3B/Protease E at 0.66, Haptoglobin-2 at 2.4 ,Protein C/Autoprothrombin IIA/Vitamin K-dependent protein C at 3.6, Serum biotinidase at 23.0, Kidney-type biotinidase at 13.0, and Valyl-tRNA synthetase/VALRS at 22.2 μg/mg of tissue protein, respectively.

HCC tissue (with PBC) has protein degradation enzymes of Ubiquitin carboxyl-terminal hydrolase isozyme L3/Ubiquitin thioesterase L3 at 3.0 μg/mg of tissue protein.

This tissue has has protein synthetic enzymes of Acylamino-acid-releasing enzyme /APH at 6.7, Aminopeptidase N/GP150 at 5.4, Serum biotinidase at 11.5, Proprotein convertase subtilisin/kexin type 6/Subtilisin/kexin-like protease PACE4 at 14.7, and Kidney-type biotinidase at 8.3 μg/mg of tissue protein, respectively.

LC tissue (named as No.6) has protein degradation enzymes of Ubiquitin-40S ribosomal protein S27a/40S ribosomal protein S27A at 0.09, Macropain subunit C9/Proteasome subunit alpha type-4; at 0.16, and Ubiquitin-activating enzyme E1/Ubiquitin-like modifier-activating enzyme 1/A1S9 protein at 1.1 μg/mg of tissue protein, respectively.

This tissue has has protein synthetic enzymes of Coagulation factor V at 3.5, Complement C2at 1.3, Insulin-degrading enzyme/Insulinase/IDE at 4.0, Serum biotinidase at 25.6, and Kidney-type biotinidase at 15.2 μg/mg of tissue protein, respectively.

HCC tissue (named as No.6) has protein degradation enzymes of Breast cancer type 1 susceptibility protein/RING finger protein 53/RING-type E3 ubiquitin transferase BRCA1 at 7.4, and E3 ubiquitin-protein ligase CBL/Proto-oncogene c-CBL at 0.055 μg/mg of tissue protein, respectively.

This tissue has has protein synthetic enzymes of 72 KD type IV collagenase/MMP-2 at 0.3, Aminopeptidase N/Microsomal aminopeptidase/GP150; at 2.6, Bifunctional glutamate/proline--tRNA ligase at 8.4, Coagulation factor V/Factor V at 5.6, Complement factor D/Adipsin at 0.21, Chymotrypsin-like elastase family member 3A/Elastase IIIA/Protease E at 0.18, Insulin-degrading enzyme/Insulinase/IDE at 13.3, Serum biotinidase at 25.4, and Kidney-type biotinidase at 9.5 μg/mg of tissue protein, respectively.

I am very grateful to Dr. Ramin Zadali (Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences, Zanjān, Iran) to leading me to these important results.　　　　

This vector is under T7 promoter so you will need IPTG to trigger the expression. Have you added the IPTG at a proper timing? How do your cells grow? Do they grow dense? How do you break the cells? Sonication? So your protein should have 6Xhis tag, do you use nickle beads to purify it?Have you try to lyse some cells, run a protein gel and perform western blot with anti-His antibody? In this way you can see in small amount whether the protein is expressed before trying to collect large amount.

It would be nice if you provide more details of the system, and method. It seems that you may load various amounts and run WB.

Heng Liang

Thank you for your response. I did all the things you mentioned, except Western Blot, but unfortunately, protein expression was not observed.

In this work:

The final concentration of IPTG is used from 0.5 to 1 mM

The cells grow at about 180 RPM and IPTG is added at a concentration of 0.6 to 0.8

I break the cells with sonication and detected the protein bond using polyacrylamide gel electrophoresis (SDS PAGE).

What strain of e coli r u using? Since cloning a eukaryotic gene then you might be facing codon usage problems

What temperature are you growing your cells? You may try growing at lower temperature. Sometimes that will help.

Have you checked that your cloned gene is in the correct reading frame? Other possibilities:

the protein is toxic to your bacteria cells

the protein forms insoluble clumps or aggregates

your IPTG isn't inducing expression - expired/wrong dose/added at wrong time

Many factors effect on cloning :

1- Vectors are accept by host or not (must be experimented) and size of gene

2- The gene integrated with host DNA or not

3- Some specific condition must be applied

If you are sure on cloning, transformation,...It may be for the wrong dose of IPTG or situation of storage of IPTG was wrong. Please used the fresh one.

Katie A Burnette

Thank you for your response.

Yes, according to the sequencing result, the gene is inserted correctly.

If the gene has a toxic effect on my bacteria, how can I eliminate this effect?

Afsaneh Sadre Momtaz

In the case of IPTG, I tested two different sources, but unfortunately, the results were similar.

If the gene is toxic, there are strategies you can try:

Article Expression of Highly Toxic Genes in E. coli: Special Strateg...

did you compare a growing curve? (with untransformed cells) sometimes the protein is toxic, and even though the promoter is not induced, its not uncommon a leak (more if you are using BL21 DE3 with T7), considering it is a relatively big protein. What method did you use to analyze expression? MAYBE, give a try extracting and concentrating with the His Tag and try a WB... to know if the construct is working at least

I have expressed 300+ proteins, both prokaryote and eukaryote, in BL21. Lack of expression can be due to just a few reasons. You have many sensible responese already. To add to that, my systematic troubleshooting process is as below:

1. Verify the DNA sequence of the expression vector. Is your insert in-frame with the his tag and the start codon? Is the promoter the right sequence? Is the ribosome binding site spaced optimally from the start codon (~7-8 bp)? Are there any frame shifts in the coding sequence of your insert? Are there rare codons for E. coli B in the coding sequence? Always codon-optimise. In 99% of cases codon optimisation and correcting errors in DNA sequence resolved the expression problems. If you can't reclone a codon optimised version of your gene then express it in Rosetta or similar strain with tRNAs for rare codons.

2. If the DNA sequence is fine, then test that the protein being expressed is not toxic. Transform a BL21AI strain with your plasmid and the expression vector with no insert as a control. BL21AI has the T7 polymerase under control of the arabinose inducible tight promoter. Compare the growth rates (A600) of control Vs your plasmid when not induced and when induced with several concentrations of arabinose. If the protein is toxic, two things can happen: (i) a large reduction in growth rate when the protein is induced or (ii) DNA rearrangements in the coding sequence or mutations in the promoter/RBS that eliminate or reduce expression - these can be determined by streaking for single colonies after induction is complete and sequencing the plasmid DNA from several colonies to check for mutations. Note that toxicity is apparent only if the protein is expressed; lack of symptoms of toxicity can also be due to zero expression in the first place. A less dramatic difference in growth rate between expressing and non-expressing cultures should be apparent even for non-toxic protein.

3. If protein is toxic to E.coli, then there are alternative strategies. (i) In the AI strain, grow to a high OD and blast with arabinose for a short time (1h) then harvest etc. (ii) add a signal sequence so that the protein is rapidly secreted into the medium (iii) Change to yeast, pichia or other expression system (iv) in vitro expression (expensive and not scalable) (v) express smaller domains of your protein (not always feasible).

Note also that 85 kDa is a large protein. In the pET/BL21 expression system, protein yield is inversely proportional to size i.e. the larger a protein the lower the yield. You may need a more sensitive detection method such as Western Blotting to see your expressed protein.

Deepan Shah Thank you for your response.

The answer was very helpful

Only I did not understand some of your answers well can you explain more about them?

1) Ribosome binding site spaced optimally from the start codon (~7-8 bp)

( I am not able to manipulate this site during gene cloning )

2) BL21AI has the T7 polymerase under control of the arabinose inducible

(I using strain bl21 DE3.

Do you mean the arabinose inducer is IPTG?

Can the method used for A1 be used for DE3?)

3) Grow to a high OD

(What OD do you suggest?)

4) Blast with arabinose for a short time (1h) then harvest etc

(Can you explain this part more?)

Thank you

Hello,

Some clarification:

1) Ribosome binding site spaced optimally from the start codon (~7-8 bp) ( I am not able to manipulate this site during gene cloning )

This is only an issue if your start codon is somewhere distal to the cloning site. 2) BL21AI has the T7 polymerase under control of the arabinose inducible (I using strain bl21 DE3. Do you mean the arabinose inducer is IPTG? The AI strain is different from BL21DE3 in that it has an arabinose-indicible T7 RNA Pol. It is induced by arabinose instead of IPTG. The advantage of this is that the ara promoter is not as leaky as the tac promoter. 3) Grow to a high OD (What OD do you suggest?)

You have to optimise this. It will depend on the growth conditions (medium) and the protein of interest. 4) Blast with arabinose for a short time (1h) then harvest etc (Can you explain this part more?) By "blast" I mean induce with arabinose for a short time. This is a good method for toxic genes.

1. Cloning: Careful check the cloned sequence, not only to check the gene sequence encoding the target protein, but also pay attention to whether the position of the target gene inserted into the vector is correct, such as whether there is a start codon, and whether the inserted position will undergo a coding shift, whether there is an enzyme cleavage site in the target sequence (if the method of homologous recombination is used, this reason is not considered)... In general, make sure that cloning is absolutely correct.

2. Induction: Whether the conditions for inducing expression are appropriate or not, temperature, time, and concentration of IPTG will affect the expression of the protein. It is suggested that you can set the gradient conditions according to these factors to conduct experiments and compare the expression under different conditions.

3. Expression: the prokaryotic expression system may lead to incorrect folding or instability of eukaryotic proteins. Methods such as codon optimization and chaperones can be tried. In addition, the molecular weight of the protein you want to express is relatively large. Generally, this series of expression vectors are suitable for expressing proteins of about 30 kDa . If you cannot see the target protein band despite the above-mentioned reasons, it is recommended to change the expression vector or even the expression system, such as the expression system of insect cells or mammalian cells.