Overview of facility usage

Protein Technology Core FacilityDeepa K.V, Thrinath.M, Bidisha Ghosh-Dutta, Narendrakumar.S, Rohini.S, Sneha.G, Sowmya K.V, Sheetal.S.K, Falguni.P, Pratibha.B.K and Muniasamy.N

Centre for Cellular and Molecular Platforms ,National Centre for Biological Sciences

GKVK Campus, Bellary Road, Bengaluru 560065, India. Phone: 080 67175101/5111

Introduction• Proteins are abundant in all organisms, acting as enzymes, antibodies, hormones or structural elements. Because of their essential role in livingsystems, great efforts have been made to elucidate their structures in order to understand their function for drug discovery and otherapplications, which includes industrial enzymes. A high level of protein expression and optimal purification is imperative for protein biologyresearch groups in academia as well as industry for broad applications.• Depending on specific applications, the system chosen for protein expression can be critical for the end point use of the desired protein.• It is a fact that each of the expression systems has its strengths and weaknesses concerning yield, proper folding, post-translationalmodification (PTM), cost, speed and ease of use. Hence, Protein technology core at C- CAMP aims to establish the following expression systemsas shown in Fig 1.• Our team at Protein technology Core (PTC), led by highly qualified experts uses cutting edge technology and works closely with the clients tooffer service and technical guidance at all steps to optimize expression and purification to generate the protein of interest. The functionalorganogram of the facility is shown in Fig 2.

Current CapabilitiesWe have established services for cloning, expression and purification of proteins in Escherichia coli and Pichia pastoris.We also offer service for gene synthesis in appropriate expression vectors.

EXPRESSION SERVICES

Expression

Platforms

Pichia

pastorisE.coli

Cell free

systems

Purification

Platforms

Affinity

tag Ion

exchangeGel filtration

HIS tag

GST tag

MBP tag Cation exchange

Anion exchange

Fig.1 : Expression and purification platforms within the facility

FacilityDirector

Customer

Facility Manager

Business Development

Cloning Expression Purification

Fig.2: Facility Organogram

STEP I

• Affinity chromatography for recombinant proteins with GST/HIS/MBP Tag

• Ion (Anion/Cation) Exchange chromatography for native proteins

STEP II

• Polishing of the final product is done by Gel Permeation or Desalting column

Pure protein

• > 95% purity is obtained with aid of Fast Protein Liquid Chromatography (FPLC) system such as ÄKTA Purifier Fig.5: Fast Protein Liquid

Chromatography (AKTA purifier)

Column before loading Column after loading Elution

Fig.6: Affinity purification of fluorescent protein mEos2 at binding, washing and elution steps using AKTA purifier.

Service workflow

Cloning

Expression

Purification

EXPRESSION PURIFICATION

CLO

NIN

G

Prof. Satyajit Mayor (NCBS)Prof. Ramaswamy (InStem)Prof. John Mercer (InStem)Dr Sudha (InStem)Dr. Vyasa Rajashekar(Fermenta Biotech)Dr Anandi K (CCAMP)Prof. Ashok Venkitaraman

Prof. Satyajit Mayor (NCBS)Prof. John Mercer (InStem)Dr. G.R.Janardhana (Mysore University)Dr Anandi K (CCAMP)Prof. Ashok Venkitaraman

Prof. Satyajit Mayor (NCBS)Dr Ashwin (NCBS)Prof. John Mercer (InStem)Dr. Tushar Kant Beuria (Institue of Life Sciences)Dr. Vyasa Rajashekar (Fermenta Biotech)

C-CAMP

NCBS

Instem

Other Institutes

Non academic

Current users Fig10: Services performedFig9: Projects delivered

Protein technology core has been successful as a small scale player with respect to protein production. Services are delivered to internal as well as external users including institutions under Bangalore biocluster (Fig.9) with regard to cloning, expression and purification of proteins as described in Fig.10.

M U T S

+ve –ve C1 C2 C3 C4

We have delivered projects to members of Bangalore Biocluster, other institutes and industries.

Fig.4: +ve – postive control (beta-galactosidase), -ve – vectorbackbone, C1-C4 selected clonesof tropomyosin for expression

Fig.3: M- Marker, U- Uninduced, T-Total, S- Soluble

The scale of operation of expression trials in E. coli and P. pastoris are currently limited to shake flasks ranging from 100ml to 1L*10 flasks (10L).

PURIFICATION SERVICES

Upcoming Services

Cell-free protein expression systemsIn general, cell-free expression system offers rapid, simple and efficient protein production as compared to other systems. E.coli based cell-free system is ideal for high throughput screening, expression of toxic proteins, protein labeling, incorporation of unusual amino acids and mutational analysis. In addition, the Leishmania tarentolae system in PTC combines the advantages of eukaryotic expression machinery with bacterial-like robustness. This project is being done in collaboration with Prof. Alexandrov Kirill from the University of Queensland, Australia.

High throughput platform (HTP)The optimization of cloning and expression in E.coli and cell-free systems is minimized by the use of a HTP system available at PTC. The Automated platforms for running PCR, agarose gel, colony picker, temperature controlled incubator shaker, and small scale purification are available in 96X2 format (Fig.11). Here, we use Gateway system (Life technologies, USA) and in-house developed recombination based ligase independent cloning (LIC) for generating expression constructs for all above systems.

Fig 12: K-Bioscience colony picker for HTP application (1200 colonies/hour)

Fig 13: Liconics 96well deep well plate shaker incubator (20 numbers of deepwellplates)

Fig.11: TECAN liquid handling workstation freedom evo 200 integrated with colony picker, two PCR machines- T ROBOT, vacuum lines for filter basedpurifications, temperature control from -10 to 70 deg*C for 96X2 well plates, shaker/incubator with ability to store >20 plates (both microtiter and deep wellplates), magnetic plate for magnetic bead based separations, 96X2 well plate reader, software for normalization of nucleic acids and proteinsamples, apparatus to run agarose gel in 96X2 well format and barcode reader for sample traceability.

Acknowledgement: S.Mohan (Business development), Sumukh Mysore (C-CAMP) , Sowjanya Kumari (C-CAMP) and Wendie (In-stem) for their constantsupport. In addition, we extend our thanks to NCBS, In-Stem and all members of C-CAMP.

• PTC capabilities include protein production using various fusion tags (GST, MBP , HIS) for assisting solubility and purification.

• The facility currently uses inducer driven (IPTG and Rhamnose) and auto-induction techniques (LacI and prBad) in twenty two different E coli strains and thirty five expression vectors for generating functional proteins.

E. coli

E. coli is a simple and commonly used host system for recombinant protein production

under different promoters

• Intracellular and extracellular expression of desirable proteins in Pichia pastoris is carried out using well defined growth media induced by methanol for over expression.

• Four different strains of Pichia pastoris and seven compatible expression vectors are available in the facility

Pichia pastoris

Pichia pastoris is eukaryotic system which facilitate simple glycosylation and formation

of correct disulfide bridges

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

3kDa to

600kDa

separation

Demonstrating CapabilitiesPost-translational modifications (N-terminal methionine acetylation) is required for the stable and functional assembly/polymerization of tropomyosin onactin. The acetylation of native protein cannot be achieved in E.coli systems but with the use of yeast based system such as P.pastoris such posttranslational modifications can be performed. To perform acetylation, the workflow is given below:

Cloning

Sequence Confirmation

Small Scale Expression

Yes

Construct from user

Re-sequencing

No

Soluble Insoluble

To Phase II

Re-folding request

Yes

Service complete

No

No

Yes

Large Scale Expression (1-10 liters)

Large Scale Purification

Purified protein (mg) provided to customer

Medium Scale Expression ( For small scale purification)

Small Scale Purification

Fig.7: Screening and confirmation (PHASE I)

Fig.8: Large scale expression and purification (PHASE II)

FlowthroughElution 2

Elution 1

~32KDa

Purified

tropomyosin

180

115

82

64

49

30

26

19

Separation of α-Tropomyosin by Anion exchangechromatography

Cloning of tropomyosin gene into pPIC3.5pichia expression vector and linearizationof the construct with SalI for integrationinto histidine gene

linear construct was electroporated intoGS115 strain. Further, they were screenedfor His+ transformants on minimal dextroseplate without histidine.

Transformed colonies were screened formut+ transformants on minimal methanolplates by comparing with mut+ and muts

controls.

Insert integration into genome confirmed bycolony PCR using insert specificprimers/vector specific primers

Small scale expression for selectedclones to find optimal post induction time.Samples were taken every 12 hours andanalyzed by SDS gel electrophoresis.

Large scale expression to obtain required quantity of protein anddownstream processing of cell paste which includes cell lysis, separationof soluble fractions, isoelectric focusing, dialysis, ion-exchangechromatography (see figure below), lyophilization and acetylation oftropomyosin was confirmed by mass spectrometric analysis..

M1 M2 U C

Lane:M1-100bp marker, M2-1Kbp

marker, U-Uncut vector, C-Cut with Sal1

mutsmut+

M: Marker 1: Positive control (pPIC3.5 contain Tropomyosin), 2: Negative control (pPIC3.5), 3 to 10: Transformants of Tropomyosin.

1-Protein Ladder, 2-Tm Positive clone, 3-Tm-positive control (beta-galactosidase), 4- Vector backbone (negative control), 5-8 are positiveclones. The same repeats in the following lanes respectively withincrement in post induction time (hours).

Positive control

SDS-PAGE to confirm purity of α-Tropomyosin