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.
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
BL21 (DE3) / Rosetta
HEK293E/F/T, Expi293, CHO-K1
Pichiapink: X33, GS115, KM71
pET15, pET22, pET28, pMAL, pET-Duet, pCDF-Duet, pGEX
pCDNA3.1, pCDNA3.4, pCMV
pAO815, pPIC series (pPIC3.5k, pPIC9k, pPICZ, pPICZa)
Vectors for each expression system
Affinity & Solubility
His-Tag HHHHHH ….
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)