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Capabilities

One-stop Technology Platform to Accelerate Drug Discovery and Development

Capabilities
  1. Capabilities
  2. Protein Engineering, Expression and Purification

Protein Engineering, Expression and Purification

HitGen’s protein science team expertise includes detailed target know-how in various target classes, gene construct design for a variety of available expression systems, various novel target proteins and multi-protein complex assembly, as well as refolding and purification skills. We focus on novel protein targets and customized Gene-to-Protein projects, whether for crystallization purposes, screening, assay development or general drug discovery research.

We produce proteins according to projects’ need and/or our clients’ specifications, starting from a gene construct designed for their purpose, with multiple tag variations and functional labelling (e.g. biotinylation), as well as defined post-translational modifications.

                                             

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Most target gene constructs are rationally designed, based on experience, literature data, and specific needs for downstream application. The purification and quality control demands are also set to meet the project requirements. HitGen’s protein expression platform includes a full suite of expression systems (E.coli, insect cells, mammalian cells, and yeast). The detailed host cell and vector information for each system, as well as the tag types frequently used are listed below.

 

 

Expression systems and host cells

E.coli

Insect   cells

Mammalian   cells

Yeast

BL21   (DE3) / Rosetta

SF9/SF21/Hi5

HEK293E/F/T,   Expi293, CHO-K1

Pichiapink:   X33, GS115, KM71

 

E.coli

pET15, pET22, pET28, pMAL,   pET-Duet, pCDF-Duet, pGEX

insect

pFastBac, pFastBac-Dual

Mammalian

pCDNA3.1, pCDNA3.4, pCMV

Yeast

pAO815, pPIC series   (pPIC3.5k, pPIC9k, pPICZ, pPICZa)

 

Affinity tags





Vectors for each expression system

Affinity

Solubility

Affinity & Solubility

Protease Site

His-Tag HHHHHH ….

SUMO

MBP

TEV ENLYFQ\S

StrepII-Tag WSHPQFEK


GST

Thrombin LVPR\GS

Flag-Tag DYKDDDDK

Fc

Ppase  LEVLFQ \ GP

 

We purify all proteins in-house on AKTA FPLC systems using the classic chromatographic methods, such as affinity chromatography, ion exchange and size-exclusion chromatography. All purified proteins can be tested with UV spectrophotometry and SDS PAGE gel analysis to ensure >90% pure. We usually produce the target proteins of interest in multi-milligram quantities. For difficult targets, we use focused small-scale screens to find suitable boundaries. We also in-house produce various tool enzymes, among which high-quality T4 DNA ligase is produced in gram quantities to meet HitGen’s internal and external requirements for DNA Encoded Library (DEL) construction.

High quality proteins can support and facilitate a number of key processes in drug discovery, and HitGen’s protein production capability feeds seamlessly into other HitGen functions/services, such as:

  • X-ray crystallography for SBDD / FBDD programs

  • In-vitro assay development

  • DNA encoded library based screening for hit identification

 

Our “crystallography grade” recombinant protein production can be available as stand-alone service or combine it with crystal structure determination.

 

 

HitGen Target Protein Production Overview

(In-house expressed target proteins cover more than 30 different classes)

 

 

 

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