General considerations

Recombinant protein expression in E. coli is often accompanied by solubility problems.

Eukaryotic proteins, which fail to stay soluble in the prokaryotic host are often found as inclusion bodies. In this work inclusion body formation of the binding modules of different cell adhesion molecules were obtained, because for the stability of the binding modules, the postranslational formation of four disulfide bonds is necessary and because standard expression strains do not have an intracellular redox potential which allows disulfide bond formation in general. Oxidative refolding of denatured protein was successful for bovine pancreatic RNAse, which was shown almost 50 years ago (e.g. HABER AND ANFINSEN, 1961). However, the presence of cysteine residues, can lead to aggregation due to wrong intramolecular and intermolecular disulfide bonds as oxidation proceeds. To find a optimal in vitro refolding procedure, several parameters, which influence the refolding experiment have to be considered.

Minimization of aggregation

The denaturing buffer contained 8 M urea, which competes with the formation of native hydrogen bonds. To avoid aggregation during the refolding experiment upon urea dilution,

B. Expression and refolding experiments of neuronal cell adhesion molecules

0.3 to 0.8 M L-arginine was added. L-arginine was successfully applied as inhibitor of aggregation in refolding experiments of Fab fragments (BUCHNERAND RUDOLPH, 1991).

Redox system

As each of the binding module consists of four Ig domains, each stabilized by an internal disulfide bond, an appropriate redox system must be found for successful in vitro refolding.

The binding modules tested here were purified under denaturing and reducing conditions to prevent wrong intramolecular and intermolecular disulfide bonds, which may result in aggregation of the target protein prior to refolding. As a starting point for the redox system, a combination of oxidized and reduced glutathione was used. Both components were shown to assisted correct disulfide bond formation of single chain Fab fragments (BUCHNERAND RUDOLPH, 1991). Initial experiments with the proteins in this work showed, that variation of the redox system is essential to obtain at least a small fraction of fully oxidized protein. Therefore, systematic variation of the redox system was carried out.

Table B.6

Combinations of reducing and oxidizing agents tested for refolding DTT

Overview of different redox systems used in refolding experiments. In parentheses are the concentration range, which was used in the respecting experiment. Each experiment covered 9 to 16 different redox conditions, which corresponded 3 to 4 different concentrations of reducing and oxidizing agent, respectively.

Refolding using dialysis

A slow removal of denaturing agent can be achieved by dialysis of the denatured sampled against a buffer, containing a lower concentration of the denaturing agent. A modification of a simple dialysis experiment is a so called controlled dialysis, where the concentration of denaturing agent in the dialysis buffer is reduced by controlled exchange of the dialysis buffer using a pump. This method was used successfully for refolding of reduced lysozyme (MAEDA ET AL., 1995). The difference between a controlled and a standard dialysis experiment is the

B. Expression and refolding experiments of neuronal cell adhesion molecules

rate of decrease of the denaturing agent, because it is proportional to the concentration difference between dialysis tube and dialysis buffer.

Refolding on a column

Affinity chromatography like IMAC (e.g. Ni-affinity chromatography) can be used to remove or dilute the denaturing agent faster than with dialysis. The protein is applied under denaturing conditions, and eluted under non denaturing conditions. A stepwise decrease in concentration of the denaturing agent or a linear decrease can be chosen. Compared to dialysis, the removal of the denaturing agent is much faster and the single molecules are separated from each other, due to their attachment to the column, which may reduce intermolecular interactions and thereby preventing aggregation during the refolding experiment.

Refolding using quick dilution

Fast removal of the denaturing agent is possible by dilution of the denatured sample with a buffer containing a lower concentration of the denaturing agent. An important parameter is a low final concentration of the target protein to avoid aggregation between folding intermediates due to hydrophobic interaction or due to intermolecular disulfide formation.

This method is advantageous in cases where a screening of different conditions is necessary, e.g. examining the effect of different components of a redox system used in different ratios and combinations (see Table B.6, page 50). Small scale experiments can be done in parallel, whereas parallelization is almost impossible with affinity chromatography and difficult with dialysis.

Modification of the target

Systematic mutations of putatively surface-expressed protein residues (glutamate and lysine residues) has been shown to improve crystallization success and crystal lattice quality (DEREWENDA, 2004). In these examples exchanges for alanine often resulted in well diffracting crystals, but yielded less soluble protein. In this work an approach was developed were single hydrophobic residues were mutated to alanine, glutamic acid, or arginine. In a first step, the three dimensional structure of the target (in this work: L1Ig1-4) was modeled using a web based server¹ (JAROSZEWSKI ET AL., 2005). According to the obtained L1Ig1-4 model, residues were selected, which have large hydrophobic side chains and were located on the surface of the model. Using the mutagenesis procedure described above, different L1Ig1-4 mutations were generated (Table B.7, page 52). The procedure worked as follows: Day 1: PCR based mutagenesis reaction and subsequent generation of transformants in E. coli strain XL10-Gold.

Day 2: inoculation of 5-10 precultures with colonies of the mutated transformants. Day 3: (a)

B. Expression and refolding experiments of neuronal cell adhesion molecules

Plasmid preparation from an aliquot (1 ml) of each preculture, and (b) expression experiments using the precultures to inoculate 20 ml volume of expression culture. A Coomassie stained gel of the expression cultures after 3 to 4 hours induction with IPTG (cultures were induced at an OD600nm of 0.8 to 2.0) showed which mutants expressed L1Ig1-4 with the predicted molecular mass (~42 kDa) and thus served as test, for positive clones. For conformation the plasmid DNA was sequenced. Expression cultures were frozen at -70 °C for further 3 to 4 hours after IPTG induction. Day 4: Isolation and purification of inclusion bodies from thawed day 3 cultures as described previously, with subsequent refolding experiments using the quick dilution method. Successfully mutated clones (verified by DNA sequencing) were used as template in subsequent mutagenesis trials to obtain L11-4 variants with several mutations in putative surface residues.

Table B.7

L1 primer used for solubility enhancement Primer name Primer sequence

L1_F95A cccactctggctccGCcaccatcacgggcaac

L1_F104A_I111A cagcaacGCtgctcagaggttccagggcGCctaccgctgc L1_M172A_I176A ggatctactggGCgaacagcaagGCcttgcacatcaagcagg L1_L243A agccgcgcctgGCcttccccaccaactcc

L1_Y290A gccgaccgtgtcaccGCccagaaccac L1_L300A cctgcagctgGCgaaagtgggcgaggaggatg

L1_L336A_L343A ctgccccgtaGCggctgcacaagccccagagccatGCatatgggagg L1_I371A acctggagaatcaacgggGCccctgtggaggagctg

L1_F104R_I111R cagcaacCGCgctcagaggttccagggcCGCtaccgctgc

L1_M172R_I176R ctccggatctactggCGCaacagcaagCGCttgcacatcaagcagg L1_L243R ggaagccgcgcctgCGCttccccaccaactcc

L1_Y290R gccgaccgtgtcaccCGCcagaaccacaacaagacc L1_L300R caagaccctgcagctgCGCaaagtgggcgaggaggatg

L1_L336R_L343R ctgccccgtaCGCgctgcacaagccccagagccatCGCtatgggagg L1_I371R acctggagaatcaacgggCGCcctgtggaggagctg

L1_F95D gccccactctggctccGATaccatcacgggcaacaac L1_F104D_I111D cagcaacGATgctcagaggttccagggcGATtaccgctgc

L1_M172D_I176D ctccggatctactggGATaacagcaagGATttgcacatcaagcagg L1_L243D ggaagccgcgcctgGATttccccaccaactcc

L1_Y290D gccgaccgtgtcaccGATcagaaccacaacaagacc L1_L300D caagaccctgcagctgGATaaagtgggcgaggaggatg

B. Expression and refolding experiments of neuronal cell adhesion molecules

Table B.7

L1 primer used for solubility enhancement Primer name Primer sequence

L1_L336D_L343D ctgccccgtaGATgctgcacaagccccagagccatGATtatgggagg L1_I371D acctggagaatcaacgggGATcctgtggaggagctg

Only those putatively surface exposed residues were considered in the solubility enhancement experiments, which do not overlap with identified natural mutations, as they are supposed to be implicated in altered L1 homophilic and heterophilic binding properties (see Figure B.3).

B. Expression and refolding experiments of neuronal cell adhesion molecules

B.2.6.2. Refolding of L1Ig1-4 and NgCAMIg1-4 - Experimental procedures