1. Coding Rules
Long time ago, we only had E. coli expression system for recombinant proteins and all catalog numbers of recombinant proteins start with CSB-RP.
Then we slowly add Yeast, Baculovirus, and Mammalian cell expression systems. In order to distinguish the catalog numbers, we rewrite the catalog numbers, EP (E.coli protein), YP (yeast protein), BP (baculovirus protein), MP (mammalian protein).
Both of CSB-RP and CSB-EP are for E. coli expressed proteins, most of them are distinguished by new catalog number and old catalog number. Only a very small part of them are distinguished by tag information and expression region. You could consult with us for specific proteins.
All catalog numbers of full-length transmembrane proteins start with CSB-CF. We could still provide partial transmembrane proteins starting with CSB-EP, CSB-YP, CSB-MP, and CSB-BP.
2. Tag Type & Tag Sequence
New Vectors containing N-terminal Tag + C myc-Tag: Updated by May 18, 2016
From now on the new orders will use the new vector, and the protein's tag will be different from before.
The N terminal His-tag/His-sumo-tag will change to N terminal His-tag/His-sumo-tag+C terminal mcy-tag, and the his will change from 6*his to 10*his.
The N-terminal GST-tag will change to N-terminal GST-tag+C terminal mcy-tag.
The N-terminal His-b2m-tag will change to N-terminal His-b2m-tag+ C terminal mcy-tag, and the his will change from 6*his to 10*his.
The proteins in stock and the proteins under production will keep as before that is 6*his. If customer still need 6*his for customized proteins, pls tell us when placing the order.
a. Complete Sequence =Tag Sequence + Target Protein Sequence (There is no other linker sequence)
b. For a given tag of different proteins, the tag sequence for the same expression system is the same.
c. The common tag information is as following:
Note: “AAAEQKLISEEDL” is the sequence of mcy-tag. “LVPRGS” is the sequence of thrombin site.
E. coli Version:
(We could provide His-tag, His-sumo-tag, GST-tag, or His-b2m-tag for E. coli at N-terminus.)
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFRT + Target Protein Sequence
MASMTGGQQMGRGSHHHHHHHHHHLVPRGSRT+ Target Protein Sequence + AAAEQKLISEEDL
MAHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGSEFRT + Target Protein Sequence
Target Protein Sequence + AAAEQKLISEEDL
RLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFRT + Target Protein Sequence
RLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHHHHHHHHHHLVPRGSRT+ Target Protein Sequence +AAAEQKLISEEDL-
MGHHHHHHIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMPKSDLEVLFQGPLGSRTHHHHHHH HHLVPRGSRT+Target Protein+ AAAEQKLISEEDL-
(We only provide His-tag for Yeast Version at N-terminus temporarily.)
EAEAYVHHHHHHEFRT + Target Protein Sequence
For Yeast version, the transformed vector is still in the verification phase, and we will still provide the tag information same as before transformation temporarily. We will inform you the tag change for Yeast version later, after finishing the verification.
(We only provide His-tag for Baculovirus Version at N-terminus.)
MHHHHHHHHHHLVPRGSRT+ Target Protein Sequence +AAAEQKLISEEDL
Mammalian cell Version:
(We only provide His-tag for Mammalian cell Version at N-terminus.)
DAAQPARRAVRSLHHHHHHHHHHLVPRGSRT+ Target Protein Sequence +AAAEQKLISEEDL
3. Frequently asked Questions and Answers about Protein Sequence and Tag Sequence
Q: 1. “The proteins in stock and the proteins under the producing will keep as before that is 6*his.”—Does it mean that Flarebio has successfully produced these proteins before, then these proteins will use old vector and won’t use new vector?
A: Yes. "The stock proteins" refer to the proteins we've successfully produced before. "The proteins under production" refer to your open orders for proteins. For these proteins, we won't use new vector.
Q: 2.How could we distinguish which are stock proteins and which are new proteins?
A: The proteins in stock protein list will use old vector. For customized proteins, we will preferentially use new vector (Except for Yeast version).
We will list specific tag information in stock protein list and will update the list regularly.
Q: 3. If there is no way for us to know, then we will continue to need to ask for all customer inquiries. This is because the Tag type is information most customers want to know in advance (pre-purchase inquiry).
A: We will proactively provide tag information for all the four versions when replying your inquiries.
Q: 4. I know activity isn’t measured – but in general –what is the impact of a given Tag type and any potential biological activity of the protein? Is a certain tag more or less likely to preserve biological activity – or does the Tag type not really make a difference? Or don’t you know?
A: Theoretically small tags generally have very small influence on protein activity. However, the specific impact on protein activity can't be concluded (There is no impact on some proteins, small impact on some proteins, and relatively great impact on some proteins).
We should conduct specific analysis for specific proteins and specific tags. In early stage, we could check related literatures. In later stage , we will mainly validate by designing control group.
We could design parallel experiment for Fusion Protein & Tag-removed Protein or Fusion Protein & Tag-protein. We haven't done relevant test for protein biological activity at present. So we can't 100% guarantee that the proteins have activity. (We will build up the detection of activity in later stage)
Q: 5. Is the new vector for all hosts – E coli, Yeast, Mammalian, Baculovirus? Or different hosts have different Tags?
A: Previously all proteins don't contain C-terminal tag. After the transformation of the vector, all proteins have myc-tag at C-terminus. Note: Yeast version is not included at present which is still in the verification phase.
The specific tag types and sequences for the four versions are listed above (E. coli: His-tag, His-sumo-tag, GST-tag; Yeast:His-tag; Baculovirus: His-tag; Mammalian cell: His-tag).
Q: 6. Standard Tag removal involve both N-terminal + C terminal Tags? For example, if customer wants Tag removal from N terminal His-tag/His-sumo-tag+C terminal mcy-tag: does that mean both the N terminal His-tag/His-sumo + C terminal myc-tag will be removed? Or what will be removed?
A: The N-terminal tag of our transformed vector is followed by a thrombin site (marked with green color above: LVPRGS). N-terminal tag can be removed by thrombin and C-terminal MYC-tag will remain. If customers have no special requirement, we will only remove N-terminal tag. If customers want to remove both tags (N terminal His-tag/His-sumo-tag + C terminal mcy-tag), we could also provide. Please consult with us when sending your inquiries.
C-terminal myc-tag removal methods are list as following:
① Directly add terminator onto downstream primer.
② Add thrombin site onto downstream primer and then remove N-terminus tag and C-terminal by enzyme digestion.
③ Connect vector before transformation.
We prefer to use the first option, only removing N-terminal tag. Because myc-tag won’t influence protein expression, and it mainly play role in myc-tag-specific antibody detection and myc-tag antibody affinity chromatography purification (generally as a second purification).
Q: 7. What do you mean by N terminal His-tag/His-sumo-tag? Please clarify. Do you mean that His-tag will change to His + C terminal myc-tag and that His-sumo-tag will change to His-sumo-tag + C terminal myc-tag?
A: Yes. It’s correct. It means that His-tag will change to His + C terminal myc-tag and that His-sumo-tag will change to His-sumo-tag + C terminal myc-tag. In short, the single label becomes the double label.
Q: 8. Why are you adding C terminal Myc-tag? For example, does it promote purification? Protein stability? Or for some other reasons?
A: We have three purposes for this tag transformation:
① The His changes from 6*his to 10*his. The main reason is that 10*His has a stronger affinity for the column material which is helpful for those proteins that can't attach to the column very well.
It is also helpful for protein purification (The specific adsorption capacity of target protein is strong, and the nonspecific adsorption ability of the impurity protein is weak, then we can design a high concentration of midazolam to further remove the impurity protein).
② We add myc-tag at C-terminus. This is mainly for the convenience of customers and us to do WB detection for myc-tag antibody, thus further test the accuracy of the target protein. Although we can also detect His-tag antibodies by WB, but relatively speaking, its specificity is not good as myc-tag.
③ We add thrombin site for all tags. It will be more convenient if customers require for tag removal later.
4. Tag Removal Service
If customer needs to remove the tag, please communicate with us in advance, otherwise, we won't remove the tag.
Not all protein tags can be removed as some proteins will be very unstable after tag removal.
If we succeed in removing the tag, we will charge for extra cost for tag removal.
If we fail in removing the tag, we won’t charge for any extra cost, and remark this information in datasheet as follows “Note: The laboratory determined that the Tag on your protein could not be removed with standard laboratory procedures. Your protein is being supplied with the Tag intact.”
Generally, the delivery time will be extended for 2-3 days.
5. Endotoxin Removal Service
If customer plans to use a protein in cell culture, please check with us if it has been endotoxin tested and if it is suitable for this purpose.
If customer has requirement for endotoxin level, please remark this information when placing the order and we could offer endotoxin removal service free of charge.
We use PMB affinity chromatography (polymyxin B-Agarose) to remove endotoxin, use LAL reagent to qualitatively detect the content of endotoxin and guarantee endotoxin level within 0.1ng / ug (1EU / ug).
Generally, the delivery time will be extended for 2-3 days.
Endotoxin removal result will be shown in COA as follows “<1.0 EU per 1μg of the protein by the LAL method.”
6. Aseptic Manufacture Processing
If customer plans to use a protein in cell culture, please check with them if aseptic manufacture processing is required. If customer has demand for aseptic manufacture processing, then please remark this information when placing the order.
We could offer aseptic manufacture processing service free of charge.
The delivery time won’t be extended.
We've performed Aseptic processing for liquid protein before lyophilization, but there may exist contamination during lyophilization process, so we can't say bacteria free for the whole process.
Aseptic manufacture processing will be shown in both of datasheet and COA as follows “0.2 μm sterile filtered 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, 50% glycerol”.
7. Western Blot Service
We could offer Western Blot service for tag antibody free of charge. If customer has demand, please this information when placing the order.
Generally, the delivery time will be extended for 2-3 days.
WB detection result will be shown in COA, but won’t be shown in datasheet.
8. Protein Purity
The purity is higher than 90% as determined by SDS-PAGE.
We adopt Nickel column affinity chromatography method for protein purification, we will decide if it needs a secondary AKTA-SEC purification by the SDS-PAGE result of the initial purity above 90%. If the initial purity SDS-PAGE result is already qualified for the QC standard (>90%), and you don’t remark any strict requirement on the purity, generally we will not conduct AKTA-SEC for secondary purification.
9. Protein Concentration
The protein concentration of each batch won't be exactly the same but we could guarantee 0.1-2.0mg/ml concentration for E. coli and Yeast, and 0.1-0.5mg/ml for Baculovirus and Mammalian cell.
If customer has special requirement for protein concentration, please communicate with us in advance.
10. Storage Conditions
For short-term storage, store at 4℃. For long-term storage, store at -20℃ or -80℃.
11. Delivery Form
The default delivery form is liquid form. If the customer has demand for lyophilized form, please remark this requirement when placing order.
Relatively speaking, the lyophilized form will be more stable than the liquid form, and the shelf life will be longer. But lyophilization may have damage to protein structure, and may affect activity of some proteins.
12. Default Storage Buffer
Tris-based buffer, PH=8.0, 50% glycerol
If customer has special requirement for the storage buffer or some components in the buffer, please remark when placing order.
13. Buffer Before Lyophilization
The buffer before lyophilization for different proteins will be different. Please ask the buffer composition each time.
The reconstitution method is the same for all proteins. Please use deionized sterile water to reconstitute proteins. (Dissolve completely. The concentration could depend on customers' experiment demand.)
It is better that concentration does not be lower than 0.1mg/ml. Proteins will be unstable if they're too diluted. Generally, we recommend 0.1-1.0mg/ml concentration. If customer has special requirements for concentration, then they could dilute the protein according to their experiment demand.
We haven’t studied whether the optimal concentration range will vary among different hosts such as E.coli, yeast, mammalian or baculovirus, so we can’t provide useful advice.
If customer will use immediately after dissolving one protein, then they don't need to add glycerol. If they won't use the protein in short term, we suggest to add some glycerol for storage.
We suggest customer to reconstitute suitable amount of protein and store the remaining protein in lyophilized powder. You could remark "The lyophilized powder should be packed into multiple vials" when placing order. The amount of lyophilized powder in each vial will depend on customer's experiment demand.
In principle, the final concentration of glycerol from 5% to 50% all will be OK. Our default final concentration of glycerol is 50%. Customers could use it as reference.
15. Protein Shelf Life
The shelf life is related to many factors, storage state, buffer ingredients, storage temperature and the stability of the protein itself.
So the shelf life of different proteins won't be exactly the same. We haven’t performed detailed research for this at present.
Relatively speaking, the lyophilized form will be more stable than liquid form, and the shelf life will be longer. But lyophilization may have damage to protein structure, and may affect activity of some proteins.
The shelf life of liquid-form protein will be 6 months around at -20℃/-80℃.
The shelf life of lyophilized form is 12 months around at -20℃/-80℃.
The shelf life of reconstituted recombinant protein is 6 months around at -20℃/-80℃. It refers that customer reconstitutes protein immediately using sterile water once receiving the item, and then add glycerol with certain final concentration, and finally store at -20℃/-80℃, and then the reconstituted protein can be stored for six months. If customers use the protein immediately, then they don't need to add glycerol, and the protein can be stored for about one week at 4℃.
The above shelf lives are concluded by our current storage experience. The specific shelf life will depend on specific protein.
Repeated freezing and thawing is not recommended. Store working aliquors at 4℃ for up to one week.
16. Frequently asked Questions and Answers for Recombinant Protein
Q: 1. Why does protein need lyophilization? What influences will the lyophilization have on proteins?
A: The proteins are very sensitive to heat, protein lyophilization can make most of the activity retain, improve protein stability and extend the storage time, meanwhile reduce shipping costs.
Lyophilization may cause partial loss of protein activity, aggregation and other degenerative problems. However, by adding protective agents (stabilizers, additives, excipients) and control ling various lyophilized conditions can reduce these negative effects as much as possible.
Tips: Generally Cusabio provides lyophilized powder, they are very stable (at least one month) at room temperature. Nevertheless, we suggest to store at -20 ℃ upon receipt of our products, to ensure 100% activity of the protein.
Q: 2. Why should we add protective agent to the protein solution before lyophilization? What’s the general protective agent? Which kind of protective agent do you usually add?
A: The protective agent is used in the process of freeze-drying and storage to protect protein. Commonly used protective agents or stabilizers include saccharides, polyols, polymers, surfactants, some proteins and amino acids etc. We usually add 8% (mass ratio by volume) of trehalose and mannitol as lyoprotectant. Trehalose can significantly prevent the alter of the protein secondary structure and the extension and aggregation of proteins during freeze-drying process; mannitol is also a universal applied lyoprotectant and fillers, which can reduce the aggregation of certain proteins after lyophilization.
Tips: For the majority of proteins, they can be only stored at 4 ℃ for a short term (about one week) after resuspended. If you want a long-term preservation, first formulated as a diluent (which must contain a carrier protein, such as 0.1% BSA, 5% HSA, or 10% FBS), then repackage and freeze at -20℃ or -80℃. Be sure to avoid repeated freezing and thawing, because each freezing and thawing cycle will cause part of the protein inactivation.
Q: 3.Why our protein products are almost invisible in pipes?
A: Cusabio protein product does not contain carrier protein or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and sucrose, and when freeze-dring the lowest salt content solution often does not form a white grid structure, but a trace amount of protein deposit within the tube, forming a thin transparent or invisible protein layer.
Tips: Before opening the lid, we recommend to centrifuge in a small centrifuge for 20-30 seconds firstly, so aggregates the protein that attached to the wall or cap of the tube in the bottom of the tube. Our quality control steps ensures that the amount of protein contained in each tube is accurate, although sometimes you can’t see the protein powder, but the protein content in the tube is still very accurate.
Q: 4. How to determine species cross-reactivity of cytokines?
A: (1) Apart from a few exceptions, most human cytokines are active on mouse cells. (2) Many mouse cytokines may also have effect on human cells, however, the activity may be lower than the corresponding human cytokines. (3) One of the few human cytokines will be more active than corresponding mouse cytokines when acting on mouse cells, such as IL-7. (4) Interferon, GM-CSF, IL-3 and IL-4 and other cytokines are species-specific and almost have no activity on non-homologous cells. (5) In contrast, fibroblast growth factor (FGF) and neurotrophin are highly conserved and both have good activity on cells of different species.
Q: 5. How to properly dissolve recombinant protein product?
A: Step 1: Centrifugate the reagent tube before opening the cap. The lyophilized powder may drift and adsorb on the tube wall or the cap due to bump during the transportation. Before opening the plastic cap, collect the lyophilized powder to the tube bottom through centrifugation, in order to completely dissolve the lyophilized powder with a small volume of liquid. Generally, need to centrifugate for 5min at 3000-3500rpm to ensure a good effect.
Step 2: Resuspend in sterile water to 0.1-1.0 mg/ml with no oscillation. This step is the dissolving step and it’s very important.
(1) Be sure to use the recommended solution to resuspend (or dissolve) the lyophilized powder. The solubility of protein is related to many factors, among them, pH and ionic strength are relatively important. The dissolving solution indicated in the product datasheet is the liquid which can completely dissolve the cytokine or recombinant protein. If the pH and ionic strength of the user's solution are inconsistent with the product datasheet, in most case, this will cause that the cytokine or recombinant protein can't be dissolved completely or even can't be dissolved, then the prepared cytokine or recombinant protein will absolutely be lack or loss of activity.
(2) Protein must be dissolved to the indicated concentration. Protein can maintain good stability within certain concentration range, which is indicated in the product datasheet, generally 0.1-1.0 mg / ml. However, the specific concentration range for different recombinant protein will be different. Above or below this concentration range, the cytokine or protein will be unstable, and it is very easy to appear the phenomenon of activity decline. Secondly, above this concentration range, it may exceed the maximum dissolved concentration of the protein, and the protein can't be dissolved completely. Furthermore, above or below this concentration range, protein aggregation may occur, which will still result in part of protein undissolved and activity decline.
(3) Must not oscillate (vortex). The oscillation mentioned here refers to the fast oscillation with vortex. Use the buffer stored at room temperature as the solvent. After adding the buffer, cover the bottle cap, gently flip the bottle by hand or place the bottle on a slowly rocking shaker, to guarantee that the buffer can touch the entire inner wall of the bottle. Place the bottle at room temperature for at least 10 - 15 minutes, then, make aliquots or use directly.
Step 3: This resuspension can be stored no more than one week at 2-8 ° C.
Resuspend the cytokines or proteins with recommended buffer to the recommended concentration according to our manual, and then store at 2-8 ° C in the refrigerator, under this condition, the activity of the cytokines or proteins can maintain up to one week. So there is enough time to do a test during 5-7 days, such as the DC induced maturation test. During the experiment period, just remove a certain amount of cytokines or proteins from the refrigerator and add into the culture medium.
In fact, the recommended concentration in our manual is a little high for usual tests. So the solution should be diluted and stored at 4 ° C, and then be used up within one week. For the dilution, please operate according to the step 4 below. Use the solution containing carrier protein to conduct dilution, otherwise, the diluted cytokines or proteins will adhere to the tube wall or bottle wall easily, which will cause the cytokines or proteins concentration decreasing, then weaken its activity sharply.
Step 4: As for long-term storage, the cytokines or proteins should be diluted further with the solution containing carrier protein (such as 0.1% BSA, 10% FBS, or 5% HAS), and then be stored at -20 ° C or -80 ° C.
If an experiment is longer than one week, or the prepared cytokines or proteins can’t be used up for one time, and then long-term storage is needed. Method: Dilute the resuspended cytokines or proteins with solution containing carrier protein (such as 0.1% BSA, 10% FBS, or 5% HAS), then aliquot and store at -20°C or -80°C, conventional refrigerator freezer or ultra-low temperature refrigerator.
Before aliquot and freeze, the resuspension should be diluted with the solution containing carrier protein. The cytokines or recombinant proteins can be diluted to any concentration, because large quantity of carrier proteins could keep the low-concentration cytokines or proteins still maintaining a high stability.
When performing the serum-free culture or animal experiment in vivo, the cytokines should contain no human or animal proteins such as BSA, FBS, HAS. As for the long-term storage, use Trehalose as the carrier, then dilute the resuspended cytokines or proteins with Trehalose solution, and then aliquot and store at -20°C or -80°C.
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