A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α

Summary

IκB Kinase 1/α (IKK1/α CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-κB transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

Abstract

A class of extracellular stimuli requires activation of IKK1/α to induce generation of an NF-κB subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-κB subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/α primarily remains associated with IKK2/β and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/α within the larger or the smaller complex pool. Constitutive activity of IKK1/α has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/α, and enable its use as a drug target, we expressed recombinant IKK1/α in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/α in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

Introduction

Transcriptional activities of the NF-κB family of dimeric transcription factors are required for diverse cellular functions ranging from inflammation and immunity to survival and death. These activities are stringently controlled in cells and a loss of regulation leads to various pathological conditions, including autoimmune disorders, and cancer^1,2,3. In the absence of a stimulus, the activities of NF-κB are kept inhibited by IκB (Inhibitor of -κB) proteins⁴. The phosphorylation of specific Ser residues on IκB proteins marks t....

Protocol

1. Preparation of Recombinant Virus Suitable for Large Scale Expression of IKK1/α

1. P1 virus preparation³²
   1. Day 1: Plate Sf9 cells (~6 X 10⁵) (passage number less than 10) in 2 mL of Sf900 III insect cell medium in each well of a 6-well plate and incubate at 27 °C. Passage cells when they reach a density of 2 to 3 X 10⁶ cells per mL in suspension by diluting into fresh media at a density of ~6 X 10⁵.
   2. Day 2: Dilute 8 µL of Sf9-transfection reagent in 100 µL of Sf900 III or Grace's Insect Medium without antibiotic and serum. Vortex briefly.

Results

Cloning and expression of different constructs of IKK1/α
Full length human IKK1/α was cloned into the baculovirus expression vector pFastBacHTa within its EcoRI and NotI restriction sites to obtain an N-terminal hexa-Histidine tagged IKK1. The tag could be removed by TEV protease digestion. Since full length IKK1/α contains flexible regions on both ends, and flexible regions usually render a protein difficult to crystallize, we cloned various truncated fragments of IK.......

Discussion

Production, crystallization and structure solution of two related IKK proteins
We set out to determine the X-ray crystal structure of IKK1/α with the notion that it would be a relatively straightforward exercise given our experience with IKK2/β protein production, crystallization, and structure determination. However, we were highly surprised that these two related proteins behaved very differently regarding the ease of crystallization. Despite efforts from several high-profile laboratori.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cellfectin/Cellfectin II	Thermo Fisher Scientific	10362100	Cellfectin is now discontinued, replaced by Cellfectin II
Sf900 III Insect cell medium	Thermo Fisher Scientific	12658-027
SF9 cells	Thermo Fisher Scientific	12659017
anti-IKK1 antibody	Novus Biologicals	NB100-56704	Previously sold by Imgenex
anti-PentaHis antibody	Qiagen	34460
PVDF membrane	Millipore	IPVH00010	Nitrocellulose can also be used
Ni-NTA agarose	Qiagen	30210
Bradford assay reagent	BioRad	500001
Superdex 200 column	GE Healthcare	28989335
Amicon concentrator	Millipore	UFC801008, UFC803008, UFC201024, UFC203024
Compound A	Bayer
Calbiochem IKK-inhibitor XII	Calbiochem	401491
Staurosporine	SIGMA	S4400
MLN120B	Millenium	Gift item
AMPPNP	SIGMA	A2647
Dextran sulfate	SIGMA	51227, 42867, 31404,
Dextran sulfate	Alfa Aesar	J62101
PEG	SIGMA	93593, 81210, 88276, 95904, 81255, 89510, 92897, 81285, 95172	Some of them are new Cat # on SIGMA catalogue. What we had was originally from Fluka that had different Cat #.
Crystallization Screens
Crystal Screen I and II (Crystal Screen HT)	Hampton Research	HR2-130
Index HT	Hampton Research	HR2-134
PEG/Ion and PEG/Ion2 (PEG/Ion HT)	Hampton Research	HR2-139
PEGRX 1 and PEGRx 2 (PEGRx HT)	Hampton Research	HR2-086
SaltRx 1 and SaltRx 2 (SaltRx HT)	Hampton Research	HR2-136
Crystal mounts	Hampton Research	HR8-188, 190, 192, 194
Synchrotron	The Advanced Photon Source (APS) at the U.S. Department of Energy’s Argonne National Laboratory	Beamline 19 ID	The Advanced Photon Source (APS) at the U.S. Department of Energy’s Argonne National Laboratory provides ultra-bright, high-energy storage ring-generated X-ray beams for research in almost all scientific disciplines.