The Production of Glycoproteins by Transient Expression in Mammalian Cells

Summary

In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, can be transfected with high efficiency (> 50% cell expression) and are amenable to culture at multi-litre scale. Growing cells in micro-plate format allows screening of large numbers of vectors in parallel to prioritise those amenable to scale-up and purification for subsequent structural or functional studies. The glycoform of the expressed protein can be modified by treating cell cultures with kifunensine which inhibits glycan processing during protein synthesis. This results in the production of a chemically homogeneous glycoprotein with short mannose-rich sugar chains attached to the protein backbone. If required, these can be readily removed by endoglycosidase treatment.

16.1 Appendix

ÄKTAxpress Glycoprotein Purification Programme

Main method:(Main)

• 0.00 Base CV 5.027 {ml} HisTrap_FF_5_ml

• 0.00 Block Set_up_System_1

  • (Set_up_System_1)

  • 0.00 Base SameAsMain

  • 0.00 ResetFracPosition

  • 0.00 PumpWash A1

  • 0.00 SystemWash 10.00 {ml}

  • 0.00 Alarm_Pressure Enabled 0.7 {MPa} 0.000 {MPa}

  • 0.00 Flow 8 {ml/min} Buffer 0.10 {ml/min} Yes

  • 0.00 ColumnPosition Position1

  • 0.00 Set_Mark “Equilibration of His Column 1”

  • 3.00 AutoZeroUV

  • 3.00 ColumnPosition Bypass

  • 3.00 Flow 0.00 {ml/min} Buffer 0.10 {ml/min} No

  • 3.00 End_Block

• 0.00 Watch_AirSensor Equal 1 Finish_load_1

  • (Finish_load_1)

  • 0.00 Base Volume

  • 0.00 InletValve A1

• 0.00 Flow 0.00 {ml/min} Buffer 0.10 {ml/min} No

• 0.00 Block Air_removal_1

  • (Air_removal_1)

  • 0.00 Base Volume

  • 0.00 InletValve A1

  • 0.00 Set_Mark “Air removal”

  • 0.00 InjectionValve Waste

  • 0.00 Flow 20 {ml/min} Buffer 0.10 {ml/min} Yes

  • 20.00 End_Block

• 0.00 Block Wash_Column_1

  • (Wash_Column_1)

  • 0.00 Base SameAsMain

  • 0.00 Set_Mark “Washing column 1”

  • 0.00 Flow 0.00 {ml/min} Buffer 0.10 {ml/min} No

  • 0.00 Watch_Off AirSensor

  • 0.00 Alarm_AirSensor Enabled

  • 0.00 Injection Valve Inject

  • 0.00 Flow 5.00 {ml/min} Buffer 0.10 {ml/min} Yes

  • 10.00 AutoZeroUV

  • 10.00 End_Block

• 0.00 Block End_fractionation_load_1

  • (End_fractionation_load_1)

  • 0.00 Base SameAsMain

  • 0.00 OutletValve WasteF1

  • 0.00 End_Block

• 0.00 Block Set_up_elution_1

  • (Set_up_elution_1)

  • 0.00 Base SameAsMain

  • 0.00 ColumnPosition Bypass

  • 0.00 PumpWash A3

  • 0.00 OutletValve LoopFracF12

  • 0.00 LoopSelection Bypass

  • 0.00 ColumnPosition Position1

  • 0.00 End_Block

• 0.00 Block Elution_Peak

  • (Elution_Peak)

  • 0.00 Base SameAsMain 5.027 {ml} HisTrap_HP_5_ml

  • 0.00 Watch_UV Greater_Than 100.000 {mAU} Peak_Collection

  • (Peak_Collection)

  • 0.00 Base Volume

  • 0.00 LoopSelection LP1

  • 0.00 Set_Mark “Peak_Start”

  • 0.00 Watch_UV Less_Than 100.000 {mAU} Peak_End

  • (Peak_End)

  • 0.00 Base Volume

  • 0.00 LoopSelection Bypass

  • 0.00 Set_Mark “Peak_End”

  • 0.00 End_Block

  • 7.50 Block Peak_End

  • (Peak_End)

  • 0.00 Base Volume

  • 0.00 LoopSelection Bypass

  • 0.00 Set_Mark “Peak_End”

  • 0.00 End_Block

  • 7.50 End_Block

  • 5.00 Watch_Off UV

  • 5.00 LoopSelection Bypass

  • 5.00 End_Block

• 0.00 Block Set_up_Gel_Filtration

  • (Set_up_Gel_Filtration)

  • 0.00 Base SameAsMain

  • 0.00 Flow 0.00 {ml/min} Buffer 0.10 {ml/min} No

  • 0.00 ColumnPosition Bypass

  • 0.00 OutletValve WasteF1

  • 0.00 PumpWash A4

  • 0.00 SystemWash 10.00 {ml}

  • 0.00 Alarm_Pressure Enabled 0.5 {MPa} 0.000 {MPa}

  • 0.00 Alarm_AirSensor Enabled

  • 0.00 Flow 1.2 {ml/min} Buffer 0.10 {ml/min} Yes

  • 0.00 End_Block

• 0.00 Block Equilibrate_Gel_Filtration

  • (Equilibrate_Gel_Filtration)

  • 0.00 Base CV 120.637 {ml} HiLoad_16/60_Superdex_75_prep_grade

  • 0.00 ColumnPosition Position5

  • 0.10 AutoZeroUV

  • 0.10 End_Block

• 0.00 Block Gel_Filtration

  • (Gel_Filtration)

  • 0.00 Base CV 120.637 {ml} HiLoad_16/60_Superdex_75_prep_grade

  • 0.00 Block Inject_Peak

  • (Inject_Peak)

  • 0.00 Base Volume

  • 0.00 InjectionValve Reinject

  • 0.00 LoopSelection LP1

  • 0.00 Set_Mark “Peak_injection”

  • 10.00 InjectionValve Inject

  • 10.00 End_Block

  • 0.30 Block Fractionation_Gel_Filtration

  • (Fractionation_Gel_Filtration)

  • 0.00 Base CV 120.637 {ml} HiLoad_16/60_Superdex_75_prep_grade

  • 0.00 OutletValve FracCollF2

  • 0.00 Fractionation 2.000 {ml}

  • 0.00 End_Block

  • 1.10 End_Block

  • 0.00 End_Method

• 0.00 Block Fractionation_load_1

  • (Fractionation_load_1)

  • 0.00 Base SameAsMain

  • 0.00 OutletValve F3

  • 0.00 End_Block

• 0.00 Block Load_Sample_1

  • (Load_Sample_1)

  • 0.00 Base Volume

  • 0.00 Flow 8 {ml/min} Buffer 0.10 {ml/min} Yes

  • 0.00 ColumnPosition Position1

  • 0.00 Set_Mark “Loading sample 1”

  • 0.00 Loop (200)#No_of_loops_for_load

  • 0.00 InletValve A2

  • (50.00)#Volume_of_part_sample InletValve A1

  • 60.00 Loop_End

  • 60.00 End_Block

• 0.00 Message “The programme finished but there is still sample!!” Screen “error”

• 0.00 End_Method