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PROTEIN EXPRESION AND PURIFICATION
Protein expression and purification is a crucial step in the projects involved in the investigation of proteins structures and functional properties. The quality of the purified protein is an essential factor. Biomedal offers services integrated in protein expression and purification, both in E. coli and yeast. Biomedal has been focused, since its fundation, on the development of news technologies that increase the efficiency of this process and the quality of the protein.
Thanks to our experience in this sector, our technology, installations, and staff, Biomedal offers you the adequate customised and flexible service with advice during all the project phases at very competitive price.
Our protein expression and purification services are grouped in three phases, enabling the customer to begin the project from the start or to make use only of the phase that is of interest.
Protein Expression and Purification in E. coli:
Expression & Purification Test:
Through this first expression and purification service, the customer obtains all the information necessary about the important data for expression and purification of the protein, such as: expression levels, solubility, purification yield, etc. These data are essential for determining the culture volume necessary to produce quality of the desired protein.
The service, as a rule of thumb, uses the CASCADETM expression system just as the purification by affinity chromatography using Biomedal´s tags, LYTAG and C-LYTAG, both included in the expression vectors of Biomedal´s catalogue and the associated reactives. If the customer has a construction with a different expression system and is interested in analyzing the initial yield, we also offer this service adapted to your necessities.
Service Specifications:
The customer contributes:
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your vector chosen with the primers necessary for its sequencing free of cost.
- Sequence of the construction with the fragment of DNA target clone.
- Information available about the important target for the project.
The service includes:
- Plasmic transformation with the cDNA insert of the problem protein in E. coli REG-12 strain.
- Induction of a small volume culture with salicylate.
- Lysis with Biomedal’s Prolyse Protein Extraction Solution, and obtaining of the soluble fraction.
- Purification with Spin-columns.
- Analysis of the results by Western blot using a specific anti-tag antibody(*).
- Analysis of the results using SDS-PAGE and Coomassie.
- Quantification of purified protein.
- Development of final technical report, including SDS-PAGE photograph, conclusions and recommendations.
With the information of the Expression and Purification Test we get data like: expression level, solubility, purification yield, etc. These data make it possible to design a specific strategy for purification and determine the volume necessary to obtain the quantity of the desired heterologous protein. So, these data allow define the more approppriate optimization service for increase yield, purity, solubility, etc. of the customer’s protein.
Complementary Services:
- Oligonucleotides synthesis service: design of specific primers of the insert for its sequencing.
- Gene cloning service: cloning of the client DNA fragment in the chosen vector.
- Sequencing service: plasmic sequencing with insert of DNA problem for verification of both cloning fragment and reading phase.
- Analysis service using Western Blot: for cases where purified protein are still observed in SDS-PAGE gel, the customer is invited to obtain additional data for design optimization.
| SERVICE | CAT. NO. |
| Western Blot (*) | SE-3346 |
| Oligosynthesis | SE-4772 |
| Gene Cloning | SE-3334 |
| Sequencing | SE-3384 - SE-3391 |
| Ref. | Product |
| SE-3338 | Protein Expression and Purification Test in E. coli |
(*) Complementary service only if the customer requires it; or for cases where purified protein in the SDS-PAGE gel is not observed.
Optimization Service:
By means of this service, Biomedal offers the possibility to optimize some factors relating to protein expression and purification such as: expression level, solubility, purity level, purification yield, etc.
Data obtained in the Western Blot analysis, carried out on the product of the previous expression and purification test as well as data provided by the client, if the latter enters the process with our service de novo, are the fundamental basis on which the appropriate optimisation strategy will be designed.
This optimization service is composed of four different modules, design to cover all the customer necessities and the different cases that could appear according to the type of protein: Specialized Strains, Conditions of Induction, Purification using Inclusion Bodies and Specific Purification.
(*) Complementary service only if the customer requires it; or for cases where purified protein in the SDS-PAGE gel is not observed.
Optimization with Specialised Strains:
This module is to design corrections for expression problems related to the metabolism of host strains. The service consists in testing expression and purification in two E. coli REG-12 strains genetically modified, alternatives to our E. coli REG-12 standart strain, these strains provide two advantages for the expression proteins:
- Better expression level for periplasmic and/or membrane proteins.
- Optimisation of codon biases due to cDNA of non-bacterial origin.
The customer will receive in the technical report the level of expression and purification obtained with each specialized strain, facilitating the choosing of the strain to use during the scaling and warning of difficulties related with this particular problem protein expression in E. coli.
Specifications:
The customer contributes (*):
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your vector chosen with the primers necessary for its sequencing free of cost.
- Sequence of the construction with the fragment of DNA clone.
The service offers:
- Plasmic transformation with the cDNA insert of the problem protein in two strains of E. coli genetically modified alternatives to our E. coli REG-12 standard strain.
- Two cultures developed in parallel (one for strain) in a small volume and induction with salicylate.
- Lysis with Prolyse Protein Extraction Solution, of Biomedal, and obtaining of the soluble fraction.
- Purification with Spin-columns.
- Analysis of the results using SDS-PAGE and Coomassie.
- Analysis of the results by Western blot analysis using a specific anti-tag antibody.
- Assessment of the purified protein.
- Development of final technical report, including SDS-PAGE photograph, conclusions and recommendations for the process of obtaining the quantity of desired protein.
(*) These requirements are only necessary if the client requests the service without having previously resorted to us for the first service of Protein Expression and Purification test.
| Ref. | Product |
| SE-4646 | Optimization with Specialised Strains |
Optimization of Induction Conditions:
This module allows the analysis of ten different conditions of induction for expression and purification of the problem protein. The customer will receive, in the form of a technical report, all the expression and purification results for the different induction experiments that were obtained with his problem protein.
Specifications:
The customer contributes (*):
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your chosen vector with the primers necessary for its sequencing free of charge.
- Sequence of the construction with the fragment of DNA clone.
The service offers:
- Design of the optimisation strategy most appropriate for each specific protein.
- Plasmic transformation with the cDNA insert of the problem protein in E. coli REG-12.
- Cultures in small volume with 10 different induction conditions.
- Lysis with Biomedal’s Prolyse Protein Extraction Solution, and obtaining of the soluble fraction.
- Purification with Spin-columns.
- Analysis of the results by SDS-PAGE and Coomassie.
- Analysis of the results by Western blot using a specific anti-tag antibody.
- Assessment of the purified protein.
- Development of final technical report, including SDS-PAGE photograph, conclusions and recommendations for the process to obtain de quantity of desired protein.
(*) These requirements are only necessary if the client requests the service without having previously resorted to us for the first service of Protein Expression and Purification test.
| Ref. | Product |
| SE-4647 | Optimization of Induction conditions |
Optimization by Purification using Inclusion Bodies:
By way of optimisation from inclusion bodies, Biomedal adjusts the purification process for those low solubility proteins and which form inclusion bodies.
The customer will send in the technical report the most appropriate method for this purification, together with the quantitative result obtained.
Specifications:
The customer provides (*):
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your vector chosen with the primers necessary for its sequencing free of charge.
- Sequence of the construction with the fragment of DNA clone.
The service offers:
- Design of the optimisation strategy most appropriate to each specific protein.
- Plasmic transformation with the cDNA insert of the problem protein in E. coli REG-12 strain.
- Cultures in small volume and induction with salicylate.
- Lysis with Prolyse Protein Extraction Solution, of Biomedal, obtaining of the soluble fraction y solubilization of the inclusion bodies**.
- Purification with specific resin**.
- Analysis of the results by SDS-PAGE and Coomassie.
- Analysis of the results by Western blot using specific anti-tag antibody.
- Assessment of purification protein.
- Development of final technical report, including SDS-PAGE photograph, conclusions and recommendations for the process to obtain de quantity of desired protein.
(*) These requirements are only necessary if the client requests the service without having previously resorted to us for the first service of Protein Expression and Purification test.
(**) Modifications of the standard procedure according to the characteristics of each specific protein.
| Ref. | Product |
| SE-4648 | Optimization by Purification using Inclusion Bodies |
Optimization by specific Purification:
This last module is perhas the most open to the concrete needs of the client in relation to the concrete aspects of purification, such as purification to a determined level (Biomedal guarantees a purity between 80 and 90% in its standard purifications), purification using alternative techniques to affinity chromatography, purification by specific tag, etc.
Specifications:
The customer provides (*):
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your chosen vector with the primers necessary for its sequencing free of charge.
- Sequence of the construction with the fragment of DNA clone.
The service offers:
- Design of the most appropriate optimization strategy to each specific protein.
- Plasmic transformation with the cDNA insert of the problem protein in E. coli REG-12 strain.
- Cultures in small volume and induction with salicylate.
- Lysis with Prolyse Protein Extraction Solution, of Biomedal and obtaining of the soluble fraction**.
- Purification with specific resine**.
- Analysis of the results by SDS-PAGE and Coomassie.
- Analysis of the results by Western blot using specific anti-tag antibody.
- Assessment of the purified protein.
- Development of final technical report, including SDS-PAGE photograph, conclusions and recommendations for the process to obtain de quantity of desired protein.
(*) These requirements are only necessary if the client requests the service without having previously resorted to us for the first service of Protein Expression and Purification test.
(**) Modifications of the standard procedure according to the characteristics of each specific protein.
| Ref. | Product |
| SE-4649 | Optimization by Specific Purification |
Protein Production:
Data obtained in the initial Expression and Purification Test service allow to the customer to evaluate the culture volume necessary to obtain the desired quantity of the heterologous protein. Additionally, if the protein has been subjected to an optimization process through one of our available modules, thi s volume will be very probably reduced, as well as the purification conditions improved, which will permit higher quality proteins yields.
However, customers who have their own constructions and data about the quantity of purified protein by volume of culture, may save time and money with our Protein Production Service for the volume corresponding to the desired quantity.
The service, as a rule of thumb, uses the CASCADETM expression system and the purification by chromatographic affinity using Biomedal´s tag, LYTAG and C-LYTAG. Both the system and the tags are included in the expression vectors of Biomedal´s catalogue and related reagents. If the customer has its own construction, with the CASCADETM system or a different system, we can offer this service adapted to your needs. Likewise, the method of purification is completely adaptable to the specific characterisitics of each protein.
Service Specifications:
The client provides (*):
- Fragment of DNA clone in a vector of Biomedal´s catalogue. Biomedal will send your vector chosen with the primers necessary for its sequencing free of charge.
- Sequence of the construction with the fragment of DNA clone
- Available information about the important target protein for the project.
The service offers:
- Plasmic transformation with the cDNA insert of the problem protein.
- Culture in specific volume for the quantity of desired protein.
- Lysis with Biomedal’s Prolyse Protein Extraction Solution and obtaining of the soluble fraction**.
- Purification with specific resin**.
- Analysis of the results in SDS-PAGE gels.
- Purified protein (a purity level between 80 and 90% is provided as standard) in the quantity requested by the client.
Complementary services:
- Oligonucleotides Synthesis Service: design of specific primers of the insert for its sequencing.
- Gene Cloning Service: cloning of the client DNA fragment in the vector chosen.
- Sequencing Service: plasmic sequencing with the insert of DNA problem for verification that the fragment cloning and reading phase are correct.
- Gene Synthesis Service: optimised design of your DNA sequence for the optimisation of its expression.
- Tag separation by specific endopeptidase service: for studies in which the polypeptidic tag tail interferes with the subsequent use of the protein.
| SERVICES | CAT. NO. |
| Optimization with Specialised Strains | SE-4646 |
| Optimization of Induction conditions | SE-4647 |
| Optimization by Purification using Inclusion Bodies | SE-4648 |
| Optimization by Specifc Purification | SE-4649 |
| Oligonucleotides Synthesis | SE-4772 |
| Gene Cloning | SE-3334 |
| Sequencing | SE-3384 - SE-3391 |
| Gene Synthesis | SE-3348 - SE-3350 |
| Tag separation by specific endopeptidase | SE-4651 |
(*) These requirements are only necessary if the client requests the service without having previously resorted to us for the first service of Protein Expression and Purification test.
(**) Modifications of the standard procedure according to the characteristics of each specific protein..
| Ref. | Product |
| SE-4652 | Protein Production in E. coli: 250 ml |
| SE-4653 | Protein Production in E. coli: 500 ml |
| SE-4654 | Protein Production in E. coli: 750 ml |
| SE-4655 | Protein Production in E. coli: 1 L |
| SE-4656 | Protein Production in E. coli: 1-3 L |
| SE-4670 | Protein Expression and Purification in E. coli + Western Blot |
2.2. Protein Expression and Purification in Yeast:
2.2.1 Expression and Purification in Saccharomyces cerevisiae:
The expression of heterologous proteins in bacterial systems is an effective way to obtain high yields of our desired protein at low cost. In some cases, yeast systems can be seen as an efficient alternative to express and purify some of these proteins.
Following the idea of the bacterial CASCADETM system, Biomedal has also developed, together with scientists of the Genetics Department of the University of Seville our own Expression System in Yeast, Y-CASCADE. Y-CASCADE technology combines the features and advantages of the well known GAL1 promoter of S. cerevisiae with the advantages of the CASCADETM system.
Y-CASCADE permits you to express your recombinant proteins in an eukaryotic expression system with a low cost inducer and no requirement changes in the media formulation during the induction of the recombinant yeast.
In addition to Y-CASCADE technology for S. cerevisiae, Biomedal, in collaboration with the Complutense University of Madrid has developed EASYEAST technology for S. cerevisiae. EASYEAST technology makes unnecessary the use of chemical, enzymatic or mechanical means to break yeast cell walls. The main advantage of this system is that intracellular proteins can be released in an easy and efficient way by growth in low osmotic media at moderate temperature (37ºC).
The technologies of Biomedal for expression in S. cerevisiae as well our technical experience allow us to offer a group of expression and purification services for this organism that are efficients, personalised, with consultancy during all the phases of the project, and at a competitive price.
The structure of the service is the same as that for E. coli, equally open to the concrete needs of the client.
| Ref. | Product |
| SE-4671 | Expression and Purification Test in S. cerevisiae |
| SE-4672 | Optimization with EASYEAST strains |
| SE-4673 | Optimization of Induction Conditions |
| SE-4674 | Optimization by Specific Purification |
Expression and Purification in Baculovirus
Expression and purification in baculovirus offers the advantages of the expression in the eukaryotic system with high levels of recombinant gene expression.
Like in the previous cases, the Biomedal service methodology in protein expression and purification in Baculovirus divided into main services:
| Ref. | Product |
| SE-4880 | Cloning into baculoviral expression vector (1 tag) |
| SE-4881 | Cloning into 2 baculoviral expression vector 2 (2 tags) |
| SE-4882 | Generation of recombinant baculovirus (1 tag) |
| SE-4883 | Generation of recombinant baculovirus (2 tags) |
| SE-4884 | Amplification of recombinant baculovirus for high titer stock |
| SE-4885 | Expression optimization |
| SE-4886 | Large scale production |
| SE-4887 | Purification of recombinant soluble proteins |
| SE-4888 | Purification of insoluble His-tagged proteins |
Expression and Purification in Mammalian
Expression and purification in baculovirus offers highest level of protein post-translational processing.
Like in the previous cases, the Biomedal service methodology in protein expression and purification in Mammalian divided into main services:
| Ref. | Product |
| SE-4803 | Cloning into mammalian expression vector |
| SE-4804 | Small scale expression test |
| SE-4808 | Medium scale production |
| SE-4810 | Purificaction of recombinant soluble proteins |
| SE-4811 | Purification of insoluble His-tagged proteins |