The human voltage dependend anion channel isoform 1 was overproduced from the pDS/RBS2 HVDAC1His6 plasmid in a modified form. The modifications affected the N terminal region, due to the plasmid construction elongated by four amino acids, as well as the Cterminal region which was extended by two linker amino acids and six histidines with respect to an effective purification and detergent exchange (Fig. 41). Figure 41: Amino acid sequence of the expressed HVDAC1.
The depicted amino acid sequence was translated from the hvdac1 gene of pDS56/RBS2HVDAC1His6 with the EXPASY translation tool TRASLATE. (http://www.expasy.ch/tools/dna.html). Differences between the natural human VDAC1 and the expressed protein are highlighted in red. Displayed secondary structure elements are depicted as predicted by the Protein Structure Prediction Server (PSIPRED; http://bioinf.cs.ucl.ac.uk/psipred/ute). Sequence variations of the generated HVDAC1 mutants are indicated by there position and amino acid alteration.
The HVDAC1 protein is comprised of 294 amino acids corresponding to a molecular weight of 32 139 Da. Calculations of the theoretical isoelectric point (pI) identifies HVDAC1 as a basic protein with a value of 9.0. This value is compared to the native channel (pIHVDAC1 = 8.6) slightly increased due
to the added amino acids. With regard to the secondary structural content a computational sequence analysis predicted 19 sheets preceded by an Nterminal located helix. The predicted sheets are evenly distributed across the region of residue 27294. About 54% of the total sequence are covered by this sheets which are comprised of 8.5 amino acids in average.
4.2 Overproduction, purification and refolding of HVDAC1 4.2.1 Overproduction of HVDAC1
All HVDAC1 samples intended for NMR structural studies had to be fully deuterated and 15Nlabelled
according to its size. Moreover, for certain NMR experiments HVDAC1 had to be 13Clabelled in
addition. The required labelling was achieved by HVDAC1 overproduction in [2H,15N]M9 and
[2H,15N,13C]M9 media, respectively. With doubling times of about 3 [h] the growth rates of E. coli in
minimal and especially in fully deuterated minimal media were quite low. Furthermore, cell growth in this media even after exhaustive D2O adaption reached only to an optical density (OD600) of 1.7 AU at
the best. Because of the low yield and the significant decreased growth rate during induction, cells were not induced before an OD600 of 0.9 AU was achieved and afterwards incubated for twelve hours.
This method allowed to obtain about 3 g wet cell mass per litre medium.
The incorporation of specifically 15N or [15N, 13C] labelled amino acids into an otherwise deuterated
HVDAC protein turned out to be more difficult. To avoid the scrambling of the 15Nlabelled amino group
from the selected to arbitrary amino acids by E. coli intrinsic aminotransferases, these samples had to be produced in a transaminase negative strain. For this reason the transaminase negative E. coli
strain DL39 was modified to contain the lacI encoding repressor plasmid [prep4] which is necessary for a tight regulation of the pDS56/RBSII based expression system. To achieve the required deuteration grade this strain had to be grown in presence of a deuterated algal lysate amino acid mixture. However, the applied algal extract naturally contains all amino acids and hence aggravates an efficient incorporation of a specific label. In order to still achieve an adequate label incorporation of at least 90%, the specifically labelled amino acids had hence to be added in tenfold excess. Because of the available quantity of the labelled amino acids this entailed, however, that in the media the applicable amount of algal extract had to be reduced to 0.1%. Under these limited conditions the constructed E. coli DL39 [prep4] strain only grew to a maximum OD600 of about 0.6 AU. The direct
expression of HVDAC1 in this media hence became impossible due to the minimum of required cell mass. To avoid this problem, the required cell mass was produced in a higher supplemented AES medium in a first cultivation step. Harvesting and washing of this culture consequently provided sufficient cell mass for the amino acid specific labeling of HVDAC in a fourfold reduced volume of algal extract limited medium. This strategy finally led to a yield of 3 g of wet cell mass per 0.5 litre of expression culture.
-3 -2 -1 0 1 2 3 4 5 op tic al d en si ty a t 6 00 n m 0.01 0.10 1.00 10.00 time [h] -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 A B C
Figure 42: Growth of E. coli M15 and DL39 HVDAC1 overproduction cultures in different media.
Cell growth was monitored by the measurement of the optical density at 600 nm. Points of IPTG addition are marked by a red line. A) E. coli M15 in TB media. B) E.coli M15 in [2H,15N,13C]M9 media. C) E.coli DL39 in 2HAES media. The grey bar indicates the time period while the cells were centrifuged,
washed and resuspended in a fourfold reduced volume of the primary 2HAES culture. The production of HVDAC1 for all other biochemical and crystallisation experiments was carried out in TB medium. In this medium the expression cultures grew normal and led to the formation of 5 g wet cell mass per litre of medium. Production of selenomethionine labelled protein led to the formation of about 2.5 g wet cell mass per litre of medium. 4.2.2 Isolation of HVDAC1 inclusion bodies
The heterologous overproduction of HVDAC1 in E. coli results in analogy to previous studies[54][53], in
the formation of inclusion bodies. Due to the HVDAC1 inclusion body formation it was possible to enrich HVDAC1 in substantial amounts by a single low spin centrifugation step after cell disruption (Fig. 43A). Although the vast majority of protein impurities could be removed by this step an additional detergent based step was applied to remove potentially attached membrane parts and hydrophobic proteins. This method led to the extraction of about 70 mg dissolved inclusion bodies from one gram of the in TB grown cells. In all other media this recovery was significantly lower but amounts up to about 25 mg inclusion bodies per gram wet cells were still present.
SDSPAGE revealed a inclusion body composition of approximately 50% HVDAC1 and several other
protein impurities (Fig. 43B). To exclude an inhomogeneous expression of HVDAC1, the nature of this
As a result it turned out that in fact only a negligible part of the impurities derived from oligomeric or processed variants of the HVDAC1. The bulk of the examined impurities belong to several other predominantly soluble proteins, often observed in inclusion body fractions.
Figure 43: HVDAC1 inclusion body (IB) purification
A) HVDAC1 IBs were isolated by three centrifugation steps (Z13) in addition to an intermediate