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Introduction
As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia very useful as a protein expression system.

SinoBio Biotech provides the following host strains for your protein expression.

SinoBio Biotech provides the following vectors for your protein expression.

Terms and timeline

Phase 1. Cloning of the target gene into P. Pichia expression vectors
    Synthesis of the PCR required primers.
    Amplification and isolation of the gene and subcloning it into P. Pichia expression vector (pPICZ£¦#945; or pPIC9K).
    Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
    Maxi-prep of the recombinant vector DNAs.
Time: 2 weeks
Provide to customers: Correct expression plasmid and sequencing result.

Phase 2. Construction of P. Pichia expression strain and optimization of the target protein expression
    Transformation of recombinant constructs into appropriate P. Pichia strain (GS115 or X33) by electroporation.
    Verification of the authenticity of the expression strain by PCR.
    Selection of P. Pichia clones for protein expression.
Time: 1 week
Provide to customers: Correct expression strain and its expression result.

Phase 3. Protein expression and purification
    1~2 liters of yeast culture will be induced and the expressed protein will be harvested by centrifugation.
    The supernatant of cell culture will be concentrated by ultrafilter.
    Protein purification by a variety of columns including affinity, gel filtration, ion exchange and hydrophobic column.
    Produce 1~10 mg target protein at soluble condition. The purity of the protein is above 80%.
    Test for the result of each step by SDS-PAGE and/or Western-blot.
Time: 3~6 weeks
1. The target protein with affinity tag (His-tag or GST-tag)
    Phase 3a. Purity of target protein>80%, do not remove tag
    Phase 3b. Purity of target protein>90%, do not remove tag
2. The target protein without affinity tag
    Phase 3c. Purity of target protein>80%
    Phase 3d. Purity of target protein>90%
Provide to customers: Qualified target protein and purification result report.

Note:
1. The soluble condition just means that the protein solution buffer does not contain any denaturant, such as urea or guanidinium hydrochloride. We do not guarantee the activity of purified protein since we can not test it.
2. According to the expression level and purification yield, we will provide you at least 1mg and at most 10mg purified protein, the price is the same.
3. Customer must provide the antibody for Western-blot if the test result is required.

Confidentiality
    Your confidential sequence to us by mail will be covered. We will sign non-disclosure or confidentiality agreements when necessary.
    A general template for MTA and CFA are available and you may change the terms according to your requirements and discuss it with SinoBio.

Delivery
    Materials delivered to customers include the following:
    1. Expression strain in glycerol.
    2. DNA sequence data (When necessary.).
    3. Required final products in conventional buffer.
    4. Manufacture reports, including the major process, interim results, quality control.
    Delivery time:
    The typical delivery time is 6 weeks. Time for shipment is about 4 business days. This may vary depending on where the customer located and accidentally, delayed by custom check.
    The final products will be shipped via fedex. DHL is optional according to the customer£¦#8217;s requirements.

Request to the Customers

    1. Tell us the target DNA sequences or Genbank accession number or protein sequence through the E-mail.
    2. Provide us the requirements of the final products essentially the purity and amount, other quality control requirements are optional with additional charge such as HPLC, AA Sequencing, MS, MS/MS, biological activity, endotoxin control etc. For details please read protein analysis.
    4. The customers need to pay for 50% fees as subscription for the first order.
    5. For more genes to be expressed or complex project, please contact us for a quote.

Why SinoBio
    Professional: SinoBio Biotech focus on recombinant protein manufacture only, and the focus makes us more professional. All of the group leaders are come from big biopharmaceuticals with more than 10 years experience on protein production. The company are well equipped with Bioengenering fermentor, AKATA, HPLC and we can provide cGMP standard for your production. The fermentation scale is from liters to 500 liters. Final production is scale from milligram to tens of gram.
    Expertise: SinoBio's protein experts have more than 300 different protein purification experience, involved in NINE biologics development, most of them are in market and some of them in clinical trails. They also developed about 40 qualified cytokines and tens of biological enzymes.
    Quality: Diverse quality control method available for your choice such as amino acid sequencing, MS, MS/MS, LAL test, HPLC in addition to conventional SDS-PAGE analysis.
    Speed: Fast turnaround time (~10 days for a basic order)
    Price: Competitive prices as low as 800$
    Confidentiality: Strictly confidential customer's data. SinoBio will sign non-disclosure or confidentiality agreements when needed.