Cysteine to Serine Substitutions in Basic Fibroblast Growth Factor: Effect on Inclusion Body Formation and Proteolytic Susceptibility During in Vitro Refolding

Abstract

We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.

Measuring and Increasing Protein Solubility

“…In the literature, this is sometimes referred to as low in vivo solubility,62 but more often, it is just called low protein solubility which can be confusing for those investigating in vitro solubility problems. Several studies have addressed in vivo solubility problems,63–67 and several excellent reviews have also been written on this subject 21, 62, 68–70. In contrast to in vitro solubility problems, in vivo solubility problems are often related to low protein stability, and hence low solubility of partially or completely unfolded protein as opposed to low solubility of folded protein (see Wilkinson and Harrison62 and references therein).…”

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

“…Being widespreadly believed that inclusion body proteins are biologically inactive and therefore useless in bioprocesses, many aggregation-prone products have been disregarded for commercialisation. Protein solubility can be tailored by either process [2] or protein [3] engineering, although most efforts have been addressed to minimize inclusion body formation by co-production of folding modulators [4], or to refold purified inclusion body proteins by chemical denaturation followed by refolding procedures [5]. Both strategies need to be adapted to particular protein species and they render largely variable results regarding the final soluble protein yield.…”

High-level expression of the xylanase from Thermomyces lanuginosus in Escherichia coli

“…To our knowledge, protein solubility can also be improved by amino acid substitutions (Dale et al 1994;Rinas et al 1992). Thus, our further work should focus on rational amino acid mutagenesis.…”